Transcription amplification system for molecular imaging

ABSTRACT

The present invention provides a transcription amplification system, comprising an effector nucleic acid molecule and a reporter molecule, which work together to produce a heterologous gene product in a cell-type, specific manner. The present invention also relates to methods for producing, detecting, imaging, and monitoring the heterologous gene product in a cell or in a subject.

This application claims priority to provisional application, U.S. Ser.No. 60/355,300, filed Feb. 8, 2002, the contents of which are herebyincorporated by reference in their entirety into this application.

Throughout this application, various publications are referenced withinparentheses. The disclosures of these publications are herebyincorporated by reference herein in their entireties.

FIELD OF THE INVENTION

The present invention relates to a transcription amplification system,comprising an effector nucleic acid molecule and a reporter molecule,which work together to produce/express a heterologous gene product in acell-type, specific manner. The present invention also relates tomethods for use of the transcription system, including methods forproducing, detecting, and/or imaging the heterologous gene product.

BACKGROUND OF THE INVENTION

Current gene therapy regimens used for treatment of cancer has manylimitations, including lack of tissue-specificity, borderline efficacy,and inadequate delivery methods. Most attempts to optimize gene therapyhave focused on delivery of the recombinant therapeutic gene to thetarget tumor and restricted expression of the therapeutic gene in thetarget tumor. Another limitation is the lack of reliable andnon-invasive methods for detecting and monitoring delivery andexpression of the therapeutic gene.

One strategy for optimizing gene therapy methods includes thetwo-component reporter gene system, or the so-called two-steptranscription amplification system (Segawa, et al., 1998 Cancer Res.58:2282-2287). In this system, the first step involves a tissue-specificor tumor-specific promoter to drive expression of a transcriptionactivator. In the second step, the transcription activator inducesexpression of a reporter or therapeutic gene product.

SUMMARY OF THE INVENTION

The present invention provides isolated, recombinant nucleic acidmolecules comprising effector sequences and reporter sequences. Theeffector and reporter molecules comprise a two-step transcriptionamplification system (TSTA) which can be used to express heterologousgene products in a cell-type specific manner. The effector and reporternucleotide sequences can be used for in vivo and in vitro production ofheterologous gene products. The present invention also provides methodsfor making and using the nucleic acid molecules having the effector andreporter nucleotide sequences.

The effector nucleotide sequences include an upstream regulatory regionwhich permits expression of the effector gene product in a cell-typespecific manner. The upstream regulatory region is operably linked to anucleotide sequence encoding a chimeric, transactivator protein whichincludes a DNA-binding domain and a transcription trans-activatordomain.

The reporter nucleotide sequences include a DNA-binding sequence and anupstream regulatory sequence. The upstream sequence can be a promoterwhich is operably linked to a heterologous gene sequence. Theheterologous gene sequence can encode a reporter gene product, atherapeutic gene product, and/or an immunologically active protein.

The chimeric transactivator protein, produced by the effector molecule,is capable of binding to the DNA-binding sequence (part of the reportermolecule) and activating transcription of the heterologous sequence.Thus, the effector gene product (e.g., the transactivator protein) iscapable of trans-activating transcription, and subsequently translation(e.g., expression), of the heterologous gene sequence.

The present invention also provides methods for producing/expressingheterologous gene products using the molecules of the TSTA system. Thefirst step includes expression of the effector gene product (e.g., thechimeric transactivator protein) in a cell-specific manner. The secondstep includes expression of the heterologous gene product. The effectorgene product causes expression of the heterologous gene product. Theheterologous gene product is produced in a cell, in a cell in a subject,or in a donor cell implanted in a subject.

The present invention provides methods for detecting production of theheterologous gene product. Such detecting methods include usingnon-invasive techniques, including positron emission tomography (PET),single photon emission computed tomography, cooled charged coupleddevice (CCD) camera optical imaging, magnetic resonance imaging,bioluminescent optical imaging, and fluorescence optical imaging.

The effector and reporter nucleotide sequences of the present inventionare DNA or RNA. Further, the invention includes nucleotides sequencesthat are identical or nearly identical (e.g., similar) with the effectorand reporter nucleotide sequences of the invention. The inventionadditionally provides polynucleotide sequences that hybridize understringent conditions to the effector and reporter nucleotide sequencesof the invention. A further embodiment provides polynucleotide sequenceswhich are complementary to the effector and reporter nucleotidesequences of the invention. Yet another embodiment provides effector andreporter nucleotide nucleic acid molecules that are labeled with adetectable marker. Another embodiment provides recombinant nucleic acidmolecules, such as a vector or a fusion molecule, including the effectorand reporter nucleotide sequences of the invention.

The present invention provides various effector and reporter nucleotidesequences, fragments thereof having essential gene activity, and relatedmolecules such as antisense molecules, oligonucleotides, peptide nucleicacids (PNA), fragments, and portions thereof.

The present invention relates to the inclusion of the effector andreporter nucleotide sequences included in an expression vector. Theexpression vector can be used to transform host cells or organisms toproduce transgenic hosts. The invention further provides host-vectorsystems and transgenic animals harboring the effector and reporternucleotide sequences. Such transgenic hosts are useful for theproduction, detection, monitoring, imaging, and visualizing theexpression of the heterologous gene product.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Shows schematic diagram of the TSTA system. The first stepinvolves the tissue-specific (e.g., PSE) expression of the GAL4-VP16fusion protein. In the second step, GAL4-VP16, in turn, drives targetgene expression under the control of GAL4 response elements in a minimalpromoter [shown are five GAL4 binding sites (bs)]. Transcription of thereporter gene, either fl or HSV1-sr39tk, leads to reporter protein,which in turn leads to a detectable signal in the presence of theappropriate reporter probe. The use of the GAL4-VP16 fusion protein canpotentially lead to amplified levels of the reporter protein (FL orHSV1-sr39TK) and therefore an increase in imaging signal.

FIG. 2: (A) TSTA system-mediated amplification of fl expression. LNCaPcells were transiently transfected in the absence (−L) and presence (+L)of androgen with (i) reporter construct alone (L5), (ii) PL (one-step),(iii) PG/L5 (two-step), and (iv) SG/L5 (control) constructs. The cellswere harvested 48 h after transfection and assayed for FL activity. Theerror bars represent SEM for triplicate measurements. (B) Cell-typespecificity of the TSTA system (fl). C6, HeLa, and LNCaP cells weretransiently transfected in the absence (−L) and presence (+L) ofandrogen with effector construct, PG, and reporter construct, L5. Thecells were harvested 48 h after transfection and assayed for FLactivity. The error bars represent SEM for triplicate measurements.

FIG. 3: (A) TSTA system-mediated amplification of HSV1-sr39tkexpression. LNCaP cells were transiently transfected in the absence (−L)and presence (+L) of androgen with (i) reporter construct alone (T5),(ii) PT (one-step), (iii) PG/T5 (two-step), and (iv) SG/T5 (control)constructs. The cells were harvested 48 h after transfection and assayedfor HSV1-sr39TK activity. The error bars represent SEM for triplicatemeasurements. (B) Cell-type specificity of the TSTA system(HSV1-sr39tk). C6, HeLa, and LNCaP cells were transiently transfected inthe absence (−L) and presence (+L) of androgen with the effectorconstruct, PG, and reporter construct, T5. The cells were harvested 48 hafter transfection and assayed for HSV1-sr39TK activity. The error barsrepresent SEM for triplicate measurements. (C) Effect of androgenconcentration on HSV1-sr39tk expression. LNCaP cells were transientlytransfected in the absence (−L) and presence (+L) of androgen with PGand T5. The androgen concentration varied from 0.1 to 10 nM. The cellswere harvested 48 h after transfection and assayed for HSV1-sr39TKactivity. The error bars represent SEM for triplicate measurements.

FIG. 4: (A) In vivo optical CCD imaging of mice carrying transientlytransfected LNCaP cells for control studies. All images shown are thevisible light image superimposed on the optical CCD image with a scalein RLU/min as shown. (Panel 1) LNCaP cells were transiently transfectedwith only the L5 vector (control) and cells implanted i.p. The animalwas then imaged after injection of D-luciferin (i.p.). (Panel 2) Thesame animal as in Panel 1 was imaged 48 h after implantation of androgenpellet by re-administering D-luciferin (i.p.). (Panel 3) LNCaP cellswere transiently transfected with the PG and L5 vectors and cellsimplanted i.p. The mouse was imaged after i.p. injection of D-luciferin.(Panel 4) The same animal imaged in Panel 3 was re-imaged in the absenceof androgen 48 h later after i.p. injection of D-luciferin. (B) In vivooptical CCD imaging of mice carrying transiently transfected LNCaP cellsfor comparison of one-step and TSTA. All images shown are the visiblelight image superimposed on the optical CCD image with a scale inRLU/min as shown. (Panel 1) LNCaP cells were transiently transfectedwith only the one-step PL vector and cells implanted i.p. The animal wasthen imaged after injection of D-luciferin (i.p.). (Panel 2) The samemouse in Panel 1 was re-imaged with i.p. injection of D-luciferin 48 hafter implantation of an androgen pellet. (Panel 3) LNCaP cells weretransiently transfected with the PG and L5 vectors (TSTA system) andcells implanted i.p. The mouse was then imaged after i.p. injection ofD-luciferin. (Panel 4) The same animal imaged in Panel 3 was re-imagedwith i.p. D-luciferin 48 h after implantation of an androgen pellet.

FIG. 5: Shows comparison of transcriptional activation in vivo using theTSTA system versus the one-step system. Shown are results of mean±SEMRLU/min in five nude mice in each group. The first group had LNCaP cellstransiently transfected with PL (one-step), and the second group hadLNCaP cells transiently transfected with PG and L5 vectors (two-step).The cells were implanted i.p. in the mice. The mice were then implantedwith androgen pellets and imaged again after 24 and 48 h. There is asignificant difference (* P<0.05) between the two groups.

FIG. 6: Components of the Chimeric TSTA System. This figure illustratesthe theory and design of the TSTA system. (A) Depiction of the two-steptranscriptional activation process. In the first step, GAL4-VP16derivatives (oval circles) are expressed in prostate cancer cells in thepresence of androgen (R1881), which activates the PSA enhancer, PSE. Inthe second step, GAL4-VP16 binds to a GAL4-responsive promoter, andactivates expression of luciferase. (B) This panel illustrates theeffector plasmids used in our analysis. We describe the detailedcomposition in Results and in Materials and Methods. The abbreviationsdenote the components: GAL4: GAL4 DNA Binding Domain. VP16 AD: VP16activation domain. Enhancer refers to the 390-bp core region bearingmultiple AREs [10]; AREI and II are proximal AREs found in the promoterdescribed by Trapman and colleagues [44]; the GAL4-VP1 to -VP4derivatives are as described [68]. E4TATA contains the adenovirus E4minimal promoter from −38 to +38 relative to the start site. (C) The FLreporters used in the analysis (described in Results). (D) The singleconstruct comprises the G5-L plasmid with the PBC-VP2 fragment insertedinto the NotI site.

FIG. 7: Comparison of Two-step vs. One-step. We grew LNCaP cells in6-well tissue culture plates and transiently transfected them with G5-Lreporter and PSE/PBC-driven GAL4-VP1 (two-step), or with luciferasereporter driven directly by PSE/PBC (one-step). We added 10 nM R1881(synthetic androgen) to the “+ligand” samples one hour aftertransfection, and measured the luciferase activities 48 hours afterstimulation. The experiments were repeated multiple times in triplicate.The measurements shown here are average values of a representativeexperiment. The vertical axis shows the relative light unit (RLU)reading from the luminometer. The error bars represent standarddeviation.

FIG. 8: Increasing the Number of GAL4 Sites and VP16 Domains AugmentsActivity. We transfected LNCaP cells with PSE-VP1 (left panel) orPBC-VP4 (right panel) and G1-, G2- and G5-L. We treated and measured thecells as in FIG. 7. The measurements shown in the two panels are from aside-by-side experiment.

FIG. 9: Comparing Different Numbers of Activation Domains. Wetransfected LNCaP cells with G5-L and either GAL4-VP1, -VP2 or -VP4expression vectors driven by the PSE or chimeric (PBC) PSAenhancer-promoter. SV40-VP4/G5-L and CMV-L serve as benchmarks. Weadjusted the DNA concentrations so that the transfections containedcomparable molar amounts of the FL gene.

FIG. 10: The Spectrum of Activities Generated by the TSTA System. Weplotted results of the previous experimental combinations side-by-sidefor direct comparison. We normalized the measurements to CMV-L activityin the presence of R1881. CMV-L was assigned a value of 1. The samplesare aligned by their activities. 1. PSE-L 2. PSE-VP1/G1-L 3.PBC-VP4/G1-L 4. PBC-L 5. PSE-VP1/G2-L 6. PBC-VP4/G2-L 7. PSE-VP1/G5-L 8.PBC-VP1/G5-L 9. PBC-VP4/G5-L 10. PBC-VP2/G5-L 11. CMV-L 12.SV40-VP4/G5-L.

FIG. 11: Cell Specific Expression and Androgen Inducibility of the TSTAsystem. (A) Immunoblot analyses demonstrating the expression GAL4-VP2from the optimized PBC-VP2 construct versus our benchmark SV40-VP2. Wetransfected LNCaP cells with PBC-VP2/G5-L or SV40-VP2/G5-L with orwithout androgen treatment. The samples were prepared, blotted andprobed with GAL4-VP16 antibodies. (B) We seeded six human cell lines insix-well plates and transfected as described. The same molar amount ofluciferase gene was transfected into all the cell types with a total DNAamount of 0.5 mg per well. We normalized all the RLU readings to thevalues obtained from the combination of SV40-VP2 and G5-L transfectedinto the same cell line. The SV40-VP2/G5-L value was set at 1 in the 3-Dplot. We employed PBC-L as a control for promoter specificity of theeffector and measured the background by transfection of G5-L alone.

FIG. 12: Cell Specific Expression of the Single Construct. Wetransfected six human cell lines with SV40-VP2/G5-L, PBC-VP2/G5-L or thesingle construct. All the cells were transfected with the same molaramount of luciferase DNA. The cells were sampled, analyzed and graphedas in FIG. 11 except we switched the axes on the cell lines andtransfected plasmids to simplify the presentation.

FIG. 13: Imaging of One-step and TSTA in Living Athymic Nude Mice.Pictures shown in the figure are bioluminescent color imagessuperimposed upon the gray-scale mouse photographs. The color scale isin units of RLU/min and is to the right of each photo. Note that thescales vary among experiments. A map representing the dorsal surface ofthe mice is on the left; the circles denote the relative position of thethree injection spots, with the transfected plasmids labeled over eachcircle. We also marked the needle points on the mouse with a red marker.A description of the group is shown on top of each panel and theacquisition time of the CCD camera for each image generated is inparentheses.

FIG. 14: Luciferase expression patterns in mice and adenoviralvector-mediated luciferase gene delivery to the liver after systemicadministration. (A) CCD images of mice injected with either AdCMV-luc orthe prostate-specific AdPSE-BC-luc via tail vein. The images for 2representative animals from each cohort at 4 days post-injection areshown. The animal designation in each cohort is indicated above theimages. BC1 and BC3 represent mouse #1 and #3, respectively, in thecohort that received AdPSC-BC-luc, and CMV4 and CMV5 indicate theAdCMV-luc injected animals. The relative light intensity (RLU/min)emitted from the animal was quantitated by computer image analysissoftware and represented by the color scale ranges from violet (leastintense) to red (most intense), shown next to the images. The maximalsignal intensity (maximum RLU/min) for each image is shown below eachimage. Liver signals in the AdCMV-luc cohort were more than 5 orders ofmagnitude higher than the AdPSE-BC-luc group. (B) Adenoviral genetransferred in livers of cohorts injected either with AdCMV-luc orAdPSE-BC-luc. Southern blot analysis of total cellular DNA, extractedfrom the livers of same animals in the 2 treatment cohorts, as in FIG.14A, after imaging at 4 days post-injection. DNA (2 and 10 μg) of eachsample was analyzed.

FIG. 15: Kinetics of transgene expression and detection of metastaticlesions in living mice bearing androgen-dependent (AD) LAPC-4 tumors.(A) Location and magnitude of luciferase expression over 3 weeks'duration after intra-tumoral injection of AdCMV-luc. The format of imagedisplay is similar to that described in FIG. 14A. However, all imagesare results of a single mouse, CMV1, monitored on sequential days afterAd injection (indicated at the top of each image). Two clear sites ofluciferase expression detection are in the liver (indicated by (1) aftersignal intensity) and in the tumor (t). Tumor signals at 15 and 21 dayspost-injection were below the minimal scale (1E+5) set. Thus, usingthese imaging parameters the signals in the tumor was unable to bedetermined (UTD). (B) The in vivo luciferase expression profile of anAdPSE-BC-luc-injected animal (BC4). The in vivo luciferase expressionwas monitored at specific days post intra-tumoral injection (listedabove the image). The signal intensity of the tumor (t) and ofextra-tumoral lesions (e) is listed below the respective images. Thesignal in the animal at 2 days post-infection, before the onset ofexpression, served as background luminescence signal (≦70).Extra-tumoral signals were detected at 21 days post-injection. (C) TheCCD images of freshly isolated organs from mouse BC4. Individual organsof BC-4 (same as FIG. 15B) were isolated and re-imaged. Due to lightsignal attenuation contributed by the covering tissues, the isolatedorgans displayed higher signal intensities than in the intact livinganimal¹².

FIG. 16: Detailed histological analysis of spinal and lung metastaticlesions. (A) Histological analysis of the spine lesion of mouse BC4.5-μm sections of spine were stained with hematoxylin/eosin (H&E) or withhuman-specific anti-cytokeratin antibody. Unmagnified sections show anelongated lesion in the mid-segment of the spine. A low-magnification(40×) micrograph showed the lesion was surrounded by muscle (Mu) andadjacent to bone (Bo). Higher magnifications (200× and 400×) of thelesion were displayed. Anti-human cytokeratin specifically stained thelesion with a characteristic intense ring of cytoplasmic staining. (B)Corresponding CCD signal and histological analysis of a spinal lesion inanother mouse BC2. Procedures for the detection and identification ofthe lesion are as described above. (C) Detection of lung metastasis byCCD imaging in another animal, and definition of the lesion by confocalmicroscopy. Low-intensity upper chest signals, right>left, were detectedin a LAPC4 AI tumor-bearing mouse (BC1) 21 days after intra-tumoralinjection of AdPSE-BC-luc under the same conditions as in FIG. 15.

FIG. 17: Expression of endogenous and exogenous androgen receptor(AR)-regulated genes in an androgen-independent (AI) prostate tumor. (A)Endogenous AR and PSA expression in AD and AI LAPC-4 tumors. The brownstaining indicates positive protein expression. PSA expression in AItumor appears to be elevated compared to the AD LAPC-4 tumor. (B) CCDimages of LAPC-4 AD or AI tumor-bearing mice 11 days after intra-tumoralinjection. The images of 3 representative animals from each cohort areshown. The AI tumors are derived from two independent tumor passages.BC3A is a different passage from BC2 and BC3. (C) AdPSE-BC-luc-mediatedluciferase expression in LAPC-4 AD and AI tumor cell suspensions afterex vivo infection. The graph displays the luciferase activity ofinfected cell extracts harvested at 3 days post-infection. Statisticalanalysis by t-test showed a significant difference (P=0.007) (two-tail).Single-cell suspension cultures were infected at 10 infectiousunits/cell and analyzed at three days post-infection⁸.

FIG. 18: Shows A schematic diagram is represented for two approaches forimaging of reporter gene expression using PET/SPECT. A reporter geneintroduced into the cell can encode for (I) an enzyme (e.g., HSV1-TK)that leads to trapping of a radiolabeled probe (left panel) or (II) anintracellular and/or extra cellular receptor (e.g., D₂R), which wouldlead to trapping of a radiolabeled ligand (right panel). In both casesif the reporter gene is not expressed, then the radiolabeled probe isnot retained.

FIG. 19: Shows MicroPET imaging of two PET reporter genes (HSV1-tk andD2R) in the same mouse using two different reporter probes (¹⁸F labeledFPCV and FESP) respectively. Specific accumulation of probes in a mousecarrying a tumor stably expressing HSV1-tk (right) and a separate tumorstably expressing D₂R (left). The accumulation of [¹⁸F]FPCV and[¹⁸F]FESP in each tumor reflects trapping due to HSV1-tk and D₂Rexpression respectively. Background signal in the intestinal tract isseen due to clearance of the probes via the hepatobiliary system. Aseparate scale for [¹⁸F]FPCV and [¹⁸F]FESP is shown (135).

FIG. 20: Shows A schematic representation of imaging reporter geneexpression by PET from a bi-cistronic vector containing an internalribosomal entry site (IRES) (left panel). Both gene A and B areco-expressed from the same vector with the help of the IRES andexpression of protein B (a PET reporter) can be imaged quantitatively bytrapping of a PET reporter probe which will give an indirect measurementof expression of gene A. MicroPET imaging of bi-cistronic geneexpression (pCMV-D₂R-IRES-sr39tk) where both the genes were imaged byusing two different PET reporter probes ([¹⁸F]FESP and [¹⁸F]FPCV) insame animal (right panel). Three C6 cell lines stably transfected withpCMV-D₂R-IRES-sr39tk and the parental C6 control cell line were injectedat 4 different sites in a single mouse. Sequential imaging of the tumorswith [¹⁸]FDG, [¹⁸F]FESP and [¹⁸F]FPCV after 10 days showed specificaccumulation of [¹⁸F]FESP and [¹⁸F]FPCV in the tumors with stablytransfected cells while accumulation of [¹⁸F]FDG is seen in all fourtumors in the FDG section image. The [¹⁸F]FDG (whole body) image showsthe mouse outline and is provided for reference. The [¹⁸F]FESP and[¹⁸F]FPCV tumor images are highly correlated, illustrating that when onemonitors one of the two genes, one can infer levels of the second gene(144).

FIG. 21: (A) Shows the nucleotide and amino acid sequence of a vectorcomprising the effector and reporter sequences (PBCVP2G5 (not Sal)). (B)Shows the nucleotide and amino acid sequence of a vector comprising theeffector and reporter sequences (PBCVP2G5-L).

FIG. 22: The adenovirus two-step transcription activation (AdTSTA)imaging system. The upper portion of the diagram shows the TSTA imagingcassette. The PSA enhancer from −4326 to −3935 (purple) was duplicated(the chimeric enhancer core) within the upstream regulatory region from−5322 to −3744 and attached to the proximal promoter from −541 to 1.Each enhancer bears a cluster of six AREs and the promoter contains twoAREs shown in blue. The PSA regulatory region is shown expressingGAL4-VP2 bearing two amino-terminal Herpes Simplex Virus 1 VP16activation domains (amino acids 413-454) fused to the GAL4 DNA bindingdomain (amino acids 1-147). A GAL4-responsive promoter is fused in thedivergent orientation to the PSA regulatory region. The GAL4-responsivepromoter contains five 17-bp GAL4 sites upstream of the adenovirus E4promoter driving firefly luciferase. The entire cassette was cloned intoa shuttle vector and introduced into Ad5 deleted (.) for E1 and E3 usingthe AdEasy™ system. The virus was propagated in 293 cells, purified andtitered. ITR Inverted terminal repeat. Bottom: simplified representationof Adenovirus serotype 5.

FIG. 23: AdTSTA activity in cancer cell lines. A. LNCaP cells wereinfected with AdTSTA at an MOI of 10 for 1 hour and treated with 10 nMR1881 (+Ligand) or vehicle (−Ligand). At the indicated time points, thecells were lysed with Reporter Lysis Buffer and firefly luciferaseactivity was analyzed either by luminometry (lower panel, Y-axis:Relative Luciferase Units/μg of total protein normalized to 100%) or byimmunoblotting (Upper panel, AR: Anti-Androgen Receptor; Tub:Anti-Tubulin; GAL4: anti-GAL4-VP16). Samples were prepared in triplicateand the average reading with standard error is shown. The triplicatesamples were mixed for immunoblot analysis. B. LNCaP, HeLa or HepG cellswere infected with AdTSTA at an MOI of 0.1 for 1 hour followed bytreatment with 10 nM R1881. The cells were harvested 48 hours later andluciferase levels were measured. Data are normalized to theligand-induced signal in LNCaP (100%). The AdCMV (MOI 0.1) signal inLNCaP is shown in hatched bars for comparison of the AdTSTA and AdCMVluciferase activity.

FIG. 24: AdTSTA specificity in animals. Left panel: 107 pfu of AdCMV orAdTSTA virus were injected through the tail vein into 3-month old maleSCID mice and imaged by CCD. The pictures show a representative mouse inthe supine position injected with AdCMV or AdTSTA on day 14. Rightpanel: LAPC9 AD xenografts were grown on the flanks of SCID mice. Whentumors reached 0.5 cm diameter tumor size, AdCMV or AdTSTA were injectedintratumorally and imaged by CCD. The picture shows representative micein the prone position on day 14. For all studies, n>3 animals were usedin each group. For each mouse, a pseudocolor image of the emitted lightis superimposed over a gray scale photograph of the mouse. A coloredbar, which indicates the intensity of the signals, is shown on the rightof the panels, with units of photons (p) acquired per second (sec) percm² per steridian (sr).

FIG. 25: Serum PSA Measurements for AD and Castrated Mice. Serum Elisameasurements of PSA at various time points during tumor growth. Valuesare in ng per ml.

FIG. 26: AR signaling in LAPC 9 tumors. SCID mice implanted with LAPC9xenografts grown to greater than 0.5 cm were injected with 107 pfu ofAdTSTA and the mice were imaged every 3-4 days until day 14.Representative mice at day 4 and day 10 post virus injection from the ADgroup, the castrated (on day 4) AD group (ADc), and the stable AI group.The bar graph summarizes a cohort of three or more and summarizes thelog of the % change in signal from day 4 (blue bars) to day 10 (purplebars). Day 4 is set at 2 (log of 100) in each case. The right panelsshow representative immunohistochemical localization of AR in thevarious tumors using anti-AR antibodies. AR stains brown against theblue-stained nuclei.

FIG. 27: Chromatin immunoprecipitation assays in LAPC9 tumors. Thediagram describes the PSA regulatory region. Short black line underneathindicates the positions targeted by PCR. Enh: enhancer core; Mid:intermediate region; Pro: proximal promoter; Ex5: PSA exon 5 (forsequence coordinates see Methods). Tumors were isolated from micesacrificed at day 10 (6 days post castration for ADc), minced,crosslinked with formaldehyde and immunoprecipitated with anti-AR. Anautoradiograph of the multiplex PCR reactions is shown for AD, AI andADc tumors. Graph of Multiple Tumor ChIP Analyses. The band intensitieswere analyzed using ImageQuant. Background values from a mock (IgG) ChIPwere subtracted from each band and normalized to the signal from onputDNA. Error bars shown are standard deviation of three values obtained inindependent experiments.

FIG. 28: ChIP analysis of RNA Polymerase II and TFIIB from LAPC9 tumors.The bands are described in the FIG. 27 legend. Representativeexperiments from AD, AI and ADc tumors are shown. The top panels are polII and the bottom panels are TFIIB. The scatter plot represent the polII binding from 4 experiments, where the ratios of pol II binding atexon 5 vs. the proximal promoter are shown.

FIG. 29: Dynamic AR signaling. A SCID mouse bearing an LAPC9 AD tumorwas injected with AdTSTA, imaged and castrated as described previously.The upper panel shows the entire time course with signals adjusted tothe same color scale. Imaging measurements and PSA levels determined bySerum ELISA are shown below the images. Lower left panel: the tumorswere extracted and subjected to immunohistological staining with ARantibodies. Lower right panel: a ChIP assay of the extracted tumors withIgG, AR and pol II antibodies.

FIG. 30: Shown are a male transgenic mouse (left) and control mouse(right) imaged with D-Luciferin using a cooled CCD Camera. The colorscale represents the relative light units for bioluminescence signal.Note the bioluminescence from the region of the prostate in thetransgenic mouse. This illustrates the use of the TSTA approach inimaging prostate specific expression of a firefly luciferase opticalreporter gene.

FIG. 31: A schematic diagram of a vector including the effector andreporter nucleotide sequences (PBCVP2G5-luc (not -sal)

FIG. 32: A schematic diagram of a vector including the effector andreporter nucleotide sequences (PBCVP2G5(L)).

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be more fullyunderstood, the following description is set forth.

The term “isolated” as used herein means a specific nucleic acid orpolypeptide, or a fragment thereof, in which contaminants (i.e.substances that differ from the specific nucleic acid or polypeptidemolecule) have been separated from the specific nucleic acid orpolypeptide.

As used herein, a first nucleotide or amino acid sequence is said tohave sequence “identity” to a second reference nucleotide or amino acidsequence, respectively, when a comparison of the first and the referencesequences shows that they are exactly alike.

As used herein, a first nucleotide or amino acid sequence is said to be“similar” to a second reference sequence when a comparison of the twosequences shows that they have few sequence differences (i.e., the firstand second sequences are nearly identical). For example, two sequencesare considered to be similar to each other when the percentage ofnucleotides or amino acids that differ between the two sequences may bebetween about 60% to 99.99%.

The term “complementary” refers to nucleic acid molecules having purineand pyrimidine nucleotides which have the capacity to associate throughhydrogen bonding to form double stranded nucleic acid molecules. Thefollowing base pairs are related by complementarity: guanine andcytosine; adenine and thymine; and adenine and uracil. Complementaryapplies to all base pairs comprising two single-stranded nucleic acidmolecules, or to all base pairs comprising a single-stranded nucleicacid molecule folded upon itself. Molecules may be fully or partiallycomplementary to the molecules of the invention.

The term “fragment” of an effector or a reporter nucleic acid moleculerefers to a portion of the effector or reporter nucleotide sequences ofthe invention. A fragment of an effector or reporter molecule, istherefore, a nucleotide sequence having fewer nucleotides than and/ormodified from the effector or reporter nucleotide sequences of theinvention.

The term “operably linked” as used herein refers to two or morenucleotide sequences which are juxtaposed so as to permit function. Forexample, a promoter sequence operably linked to a nucleotide sequenceencoding a gene product refers to the promoter sequence juxtaposed tothe nucleotide gene sequence so that the promoter is capable ofcontrolling the transcription of the gene sequence.

Effector Sequences

The present invention provides effector nucleic acid molecules includingan upstream regulatory sequence operably linked to a nucleotide sequenceencoding a chimeric, transcription activator protein (e.g.,transactivator protein). The upstream regulatory sequence can includeone or more of promoters and/or enhancers or any combination thereof.

The enhancer or promoter sequences can include portions havingfunctions, such as nucleotide sequences that are known to bind cellularproteins or agents. The enhancer or promoter sequence function tomediate transcription of any operably linked gene sequence. Theseportions can be arranged within the enhancer or promoter sequences in awild-type arrangement, or in a modified arrangement. For example, amodified enhancer or promoter sequence can include repeat sequences of aparticular portion that bind cellular proteins or agents. Alternatively,the modified enhancer or promoter sequence can include repeat sequencesof two or more portions. The repeat sequences can be arranged in ahead-to-head or in a head-to-tail orientation. The cell-specific,upstream regulatory sequence can be intact (e.g., wild-type) or modifiedto include insertions, deletions, substitutions, or mutations.

The regulatory sequence can be a cell-specific regulatory sequenceisolated from various cell types including: blood, prostate, brain,lung, stomach, bladder, pancreas, colon, breast, ovary, uterus, cervix,liver, muscle, skin, or bone.

The regulatory sequence can be isolated from various tissue-specificgenes, including transferrin, tyrosinase and tyrosinase-related genes(melanocytes), albumin (liver), muscle creatinine kinase (muscle),myelin basic protein (oligodendrocytes and glial cells), glialfibrilllary acidic protein (glial cells), and NSE (neurons).

The tumor-specific, regulatory sequence can be isolated from varioustumor-specific genes, including VEGF, KDR, E-selectin, endoglin, AFP(liver tumor), CEA (various adenocarcinomas), erbB2 (breast andpancreatic cancer), muc-1(DF3) (breast cancer), ALA (breast cancer), BLG(breast cancer), osteocalcin (osteosarcoma and prostate cancer), SLP1(ovarian and cervical cancer), HRE (solid tumors), Grp78(BIP) (solidtumors), L-plastin (cancer cells), and hexokinase II (cancer cells).

The regulatory sequences can be isolated from treatment responsive genes(egr-1, t-PA, mdr-1, hsp70) or from cell cycle-regulated genes (E2F-1,cycline A, and cdc25C). See Nettelbeck for a review (Trends in Genetics(2000) 16:174-181).

The regulatory sequences can be from any viral source, includingadenvirus, adenovirus-associated virus, retrovirus, and lentivirus.

The regulatory sequences can be constitutive or inducible. Aconstitutive regulatory sequence includes CMV (cytomegalovirus), SV40(Simian virus) and RSV (rous sarcoma virus), yeast beta-factor, alcoholoxidase and PGH.

The regulatory sequences can be inducible which are regulated byenvironmental stimuli or the growth medium of the cells, including thosefrom the genes for heat shock proteins, alcohol dehydrogenase 2,isocytochrome C, acid phosphatase, enzymes associated with nitrogencatabolism, and enzymes responsible for maltose and galactoseutilization.

In one embodiment, the cell-specific, upstream regulatory sequence caninclude a Prostate Specific Antigen (PSA) promoter (Pang, et al., 1995Hum. Gene Ther. 6:1417-1426; Lee, et al., 1996 Anticancer Res.16:1805-1811; Martiniello, et al., 1998 Hum. Gene Ther. 9:1617-1626;Pang, et al., 1997 Cancer Research 57:495-499; L Wu, et al., 2001 GeneTherapy 8:1416-1426).

In another embodiment, the cell-specific regulatory sequence can includea Prostate Specific Cell Antigen (PSCA) promoter (Watabe, et al., 2002Proc. Natl. Acad. Sci. USA 99:401-406; Bahrenberg, et al., 2001 CancerLett. 168:37-43; Jain, A., Lam, A., Carey, M. and Reiter, R. (2002)Indentification of an Androgen-Dependent Enhancer Within the ProstateStem Cell Antigen Gene. Molecular Endocrinology 16:2323-2337.).

In another embodiment, the cell-specific regulatory sequence can includea human Kallikrien 2 (hK2) promoter (Song, et al., 1996Immunopharmacology 32:105-107; Song, et al., 1997 Human Genet.99:727-734; Wang, et al., 1997 Immunopharmacology 36:221-227; Chao andChao 1997 Immunopharmacology 36:229-236; Xiong, et al., 1997 Biochem. J.325(Pt 1):111-116; Zhang, et al., 1999 Endocrinology 140:1665-1671; Xie,et al., 2001 Hum Gene Ther. 12:549-561).

The cell-specific, upstream regulatory sequence can be isolated fromvarious organisms including prokaryote or eukaryote organisms, such asbacteria, virus, yeast, plant, insect, or mammal. The mammals includemouse, rat, rabbit, dog, cat, horse, cow, goat, pig, fish, monkey, apeor human. In one embodiment, the upstream regulatory sequence is anintact, human PSA enhancer sequence including nucleotides −5824 through−2855, linked to an intact human PSA promoter sequence including −541through +12.

The upstream regulatory sequence is operably linked, to controltranscription of the nucleotide sequence encoding a transactivatorprotein. The transactivator protein includes a DNA-binding domain,linked in-frame, with a transcription activation domain. The DNA-bindingdomain can include the DNA-binding domain of yeast GAL4, or bacterialLexA The transcription activation domain can include the transactivatordomain of herpes simplex VP-16 (Sadowski, et al., 1988 Nature335:563-564) or forkhead in rhabdomycosarcoma (FKHR) (Massuda, et al.,1997 Proc. Natl. Acad. Sci. USA 94:14701-14706). Other transcriptionalactivator domains can be used (Ma and Ptashne 1987 Cell 48:847-853; Maand Ptashne 1987 Cell 51:113-119; Brent and Ptashne 1985 Cell43:729-736; Hope and Struhl 1986 Cell 46:885-894 (e.g., GCN4); Gill andPtashne 1987 Cell 51:121-126). Other transcriptional activator domainsinclude MAPK-responsive ELK-1, steroid receptors, and those from the Etsfamily.

In one embodiment, the effector sequence includes between 1 through 4copies of VP-16. In another embodiment, the nucleotide sequence encodingthe transactivator protein includes a DNA-binding domain of yeast GAL4and between 1 through 4 copies of a transactivator domain of herpessimplex VP-16 (Emami and Carey 1992 The EMBO Journal 11:5005-5012).

In a preferred embodiment, the effector nucleotide sequence includes: anintact upstream regulatory sequence of a human PSA enhancer sequenceincluding nucleotides −5824 through −2855, linked to an intact human PSApromoter sequence including −541 through +12, operably linked to anucleotide sequence encoding the transactivator protein includes aDNA-binding domain of yeast GAL4 and a transactivator domain of herpessimplex VP-16.

In a more preferred embodiment, the effector nucleotide sequenceincludes: a modified upstream regulatory sequence including two copiesof a human PSA enhancer sequence including nucleotides −4326 through−3935 (Pang, et al., 1997 Cancer Res. 57:495-499), with an intervening890 bp sequence from −3743 through −2855 between the enhancer andpromoter deleted, linked to an intact human PSA promoter sequenceincluding −541 through +12, operably linked to a nucleotide sequenceencoding the transactivator protein includes a DNA-binding domain ofyeast GAL4 (encoding amino acids 1-147) and between 1 through 4 copiesof a transactivator domain of herpes simplex VP-16 (encoding amino acids413-454).

The most preferred embodiment of the effector nucleotide sequenceincludes 2 copies of the transactivator domain of VP16, encoding aminoacids 413-454.

Reporter Sequences

The present invention provides reporter nucleotide sequences including 1through 9 copies of a DNA-binding sequence. In one embodiment, theDNA-binding sequences can be isolated from yeast GAL4 or bacterial LexAor from PAX3 paired box gene 3 (PAX3) (Massuda, et al., 1997 Proc. Natl.Acad. Sci. USA 94:14701-14706). The DNA-binding sequence is linked to apromoter sequence. The promoter sequence can be a minimal promotersequence. The promoter sequence includes sequences necessary for corepromoter function. The promoter sequence can include a TATA boxsequence. The promoter sequence can be isolated from any sourceincluding bacteria, virus, yeast, plant, insect, or mammal. Inparticular, the minimal promoter can be isolated from any virusincluding adenovirus, adenoviral-associated, retrovirus, lentivirus,herpes simplex virus, or retrovirus. In one embodiment, the promoter isisolated from any serotype of adenovirus, including serotypes A throughF. In another embodiment, the promoter is isolated from any adenoviralearly region, including E1a, E1b, E2a, E2b, E3, and E4. In a preferredembodiment, the promoter includes the TATA box sequence from adenoviralE4 gene.

In reporter nucleotide sequence invention includes a promoter operablylinked to a heterologous nucleotide sequence encoding a heterologousgene product. In one embodiment, the reporter nucleotide sequenceincludes a minimal promoter operably linked to a chimeric heterologousgene sequence encoding two or more different heterologous gene products.In this embodiment, the different heterologous gene sequences are joinedto form a fusion sequence which is operably joined in-frame, or thedifferent heterologous sequences are arranged in a bi-cistronicconstruct (141-143). Other constructs include a multiple promoterconstruct where the different heterologous sequences are joined to adifferent promoter sequence. The reporter nucleotide sequence includes abi-directional promoter sequence operably linked to two differentheterologous gene sequences (148). Alternatively, two or more differentreporter nucleotide sequences can be introduced to a cell, where thedifferent reporter nucleotide sequences comprise a promoter operablylinked to a heterologous nucleotide sequence encoding a heterologousgene product.

The heterologous gene product can encode a reporter gene product, atherapeutic gene product, and/or an immunologically active proteincomprising a domain that binds a target.

The reporter gene product includes proteins that are detectable viavarious methods including colorimetric, fluorescence, optical,biochemical assays, or radiotracing. The reporter gene sequence canencode beta-galactosidase, luciferase, chloramphenicolacetyl-transferase, dopamine type-2 receptor (D₂R) or mutant formsincluding D₂R80A and D₂R194A, and somatostatin receptor type-2 (SSTR2),or beta-lactamase. The reporter gene sequence can encode greenfluorescent protein (GFP), or derivatives thereof such as YFP (yellowfluorescent protein), CFP (cyan fluorescent protein) or RFP (redfluorescent protein).

Alternatively, the heterologous gene sequence encodes proteins havingtherapeutic function such as cytotoxic function. The heterologous geneproduct having therapeutic function includes, but is not limited to, atoxin, a cytokine, an antibody, lymphokine, oncostatin, enzyme, or anenzyme that converts a prodrug into a cytotoxic drug.

Examples of therapeutic gene products include, but are not limited toricin, ricin A-chain, diphteria toxin, Pseudomonas exotoxin (PE) A,PE40, abrin, arbrin A chain, modeccin A chain, alpha-sarcin, gelonin,mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin,calicheamicin, sapaonaria officinalis inhibitor, or maytansinoids.

Therapeutic gene products which convert a prodrug into a cytotoxic drug(e.g., pro-drug activating enzymes), including thymidine kinase(Oldfield 1993 Hum. Gene Therapy 4: 39-69). Thymidine kinase convertsprodrugs acyclovir (9-((2-hydroxyethoxy)methyl)guanine)) or its analogganciclovir (9-((2-hydroxy-1-(hydroxymethyl)ethoxy)methyl)guanine) intointermediates capable of inhibiting DNA synthesis of a cell. Cells thatexpress thymidine kinase are susceptible to cell killing uponadministration of the prodrug.

Other examples of therapeutic gene products that convert a prodrug intoa cytotoxic drug include cytosine deaminase (Huber 1993 Cancer Res. 53:4619-4626), nitroreductase (Green 1995 British J. Surgery 82: 1546),carboxypeptidase A (Hamstra 1999 Hum. Gene Therapy 10:235-248);linmarase (Cortes 1998 Gene Therapy 5:1499-1507); xanthine-guaninephospho-ribosyltransferase (Mrox 1993 Hum. Gene Therapy 4:589-595);beta-lactamase (Shepard 1991 Bioorg. Med. Chem. Lett. 1:21-26); alkalinephosphatase (Senter 1988 Proc. Natl. Acad. Sci. 85:48424846);carboxypeptidase G2 (Bagshawe 1991 Dis Markers 9:233-238); DT diaphorase(Knox 1993 Cancer Metasta. Rev. 12:195-212); and beta-glucuronidase(Roffler 1991 Biochem. Pharmacol. 42:2062-2065).

Other examples of pro-drug activating enzymes include mutant forms ofthe pro-drug activating enzymes. A mutant form of thymidine kinase,HSV1sr-39TK) utilizes ganciclovir and penciclovir substrates effectivelythan thymidine compared to wild type thymidine kinase (Gambhir 2000Proc. Natl. Acad. Sci. 97:2785-2790).

The heterologous sequence can be a nucleotide sequence encoding animmunologically active protein comprising a domain capable of binding atarget. The immunologically active protein can be a single-chain,immunologically active protein. The domain can include anantigen-binding site of an antibody that binds the target. The proteindomain can include at least one complement determining region of anantibody that binds the target.

The target can be a target protein, a cell-surface antigen, or acell-surface receptor. For example, the target protein can betyrosinase, tyrosinase-related protein, albumin, muscle creatininekinase, myelin basic protein, glial fibrilllary acidic protein, NSE,KDR, E-selectin, endoglin, AFP, CEA, erbB2, muc-1 (DF3), ALA, BLG,osteocalcin, SLP1, HRE, Grp78(BIP), L-plastin, and hexokinase II, egr-1,t-PA, mdr-1, hsp70, E2F-1, cycline A, or cdc25C).

The immunologically active protein can bind to a target protein,including PSA, PSCA, or Kallikrien 2 (hK2).

The methods for producing the immunologically active proteins of theinvention, synthesis of oligonucleotides, PCR, transforming cells,constructing vectors, expression systems, and the like, are well knownin the art (Sambrook et al., eds., Molecular Cloning, A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory Press (1989); U.S.Pat. No. 5,637,481 issued to Ledbetter et al).

The nucleotide sequences encoding the immunologically active protein maybe expressed in a variety of systems. An expression vector, encoding theimmunologically active protein is introduced (e.g, transformed) intosuitable host cells, such as a bacterial cell (Chaudhary et al., 1987Proc. Natl. Acad. Sci. USA 84:4538-4542; Cohen, 1971 Proc. Natl. Acad.Sci. USA (1972) 69:2110). The expression vector may be introduced intomammalian host cells using DEAE-dextran mediated transfection, calciumphosphate co-precipitation, lipofection, electroporation, protoplastfusion, or other methods known in the art including: lysozyme fusion orerythrocyte fusion, scraping, direct uptake, osmotic or sucrose shock,direct microinjection, indirect microinjection such as viaerythrocyte-mediated techniques, and/or by subjecting host cells toelectric currents. The above list of transfection techniques is notconsidered to be exhaustive, as other procedures for introducing geneticinformation into cells will no doubt be developed.

The production of the immunologically active protein can be detectedusing Coomassie stained SDS-PAGE and/or immunoblotting usinganti-idiotypic antibodies that bind to the immunologically activeprotein.

The heterologous gene product, including the reporter gene product, thetherapeutic gene product, or the immunologically active protein, can bejoined to a cytotoxic agent thereby forming a conjugated molecule.

Techniques for conjugating or joining cytotoxic agents to antibodies arewell known and can be adapted for joining cytotoxic agents to theheterologous gene products (see, e.g., Arnon et al., “MonoclonalAntibodies For Immunotargeting Of Drugs In Cancer Therapy”, inMonoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For DrugDelivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al.(eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in MonoclonalAntibodies '84: Biological And Clinical Applications, Pinchera et al.(eds.), pp. 475-506 (1985); and Thorpe et al., “The Preparation AndCytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev.,62:119-58 (1982); Vitetta, E. S. et al., 1993, Immunotoxin therapy, inDeVita, Jr., V. T. et al., eds, Cancer: Principles and Practice ofOncology, 4th ed., J.B. Lippincott Co., Philadelphia, 2624-2636).

Examples of cytotoxic agents include, but are not limited to ricin,ricin A-chain, doxorubicin, daunorubicin, taxol, ethiduim bromide,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,dihydroxy anthracin dione, actinomycin D, diphteria toxin, Pseudomonasexotoxin (PE) A, PE40, abrin, arbrin A chain, modeccin A chain,alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin,curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor,maytansinoids, and glucocorticoid and other chemotherapeutic agents, aswell as radioisotopes such as ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

The heterologous gene products, including the reporter gene product, thetherapeutic gene product, or the immunologically active protein, can bejoined to a detectable label, including a radioisotope, a fluorescentcompound, a bioluminescent compound, chemiluminescent compound, a metalchelator or an enzyme.

Recombinant Nucleic Acid Molecules

Also provided are recombinant DNA molecules that include the effectorand/or the reporter nucleic acid molecules of the invention, or afragment thereof. As used herein, a recombinant DNA molecule is a DNAmolecule that has been subjected to molecular manipulation in vitro.Methods for generating rDNA molecules are well known in the art, forexample, see Sambrook et al., Molecular Cloning (1989). In the preferredrecombinant DNA molecules of the present invention, the effector and/orthe reporter sequences, or fragments thereof, are operably linked to oneor more expression control sequences and/or vector sequences.

Vectors

The nucleic acid molecules of the invention may be recombinant moleculeseach comprising the sequence, or portions thereof, of an effector ofreporter nucleotide sequence. The term vector includes, but is notlimited to, plasmids, cosmids, and phagemids. The effector and reporternucleotide sequences can be joined to a single vector or to twodifferent vectors. The effector and reporter nucleotides sequences canbe arranged in a head-to-head or head-to-tail orientation on a singlevector.

The vector of the invention can be an autonomously replicating vectorcomprising a replicon that directs the replication of the recombinantDNA within the appropriate host cell. Alternatively, the vector directsintegration of the recombinant vector into the host cell. Various viralvectors may be used, such as, for example, a number of well knownretroviral and adenoviral vectors (Berkner 1988 Biotechniques6:616-629).

The vectors include, for example, a vector derived from herpes simplexvirus that targets neuronal cells (Battleman et al., J. Neurosci.13:941-951 (1993), an immunodeficiency virus that targets hematopoieticcells (Carroll et al., J. Cell. Biochem. 17E:241 (1993), and recombinantadeno-associated viral vectors having general or tissue-specificpromoters (Lebkowski et al. U.S. Pat. No. 5,354,678). Recombinantadeno-associated viral vectors can be used to integrate the nucleic acidmolecules of the invention into the genome of a cell (Lebkowski et al.,Mol. Cell. Biol. 8:3988-3996 (1988).

Retroviral vectors can be used to produce the heterologous gene product.These vectors have broad host and cell type ranges, integrate intorandom sites in the host genome, express genes stably and efficiently,and under most conditions do not kill or obviously damage their hostcells.

Retroviral vectors contain retroviral long terminal repeats (LTRs) andpackaging (psi) sequences, as well as plasmid sequences for replicationin bacteria and may include other sequences such as the SV40 earlypromoter and enhancer for potential replication in eukaryotic cells.Much of the rest of the viral genome is removed and replaced with otherpromoters and heterologous gene sequences. Modified (defective)retroviruses can be made in which at least one of the genes required forreplication is replaced by the gene to be transferred. Vectors arepackaged as RNA in virus particles following transfection of DNAconstructs into packaging cell lines. These include psi2 which produceviral particles that can infect rodent cells and psiAM and PA 12 whichproduce particles that can infect a broad range of species. Methods ofpreparation of retroviral vectors have been described (Moolten & Wells,J. Natl. Cancer Inst., 82:297-300 (1990); Wolff et al., Proc. Natl.Acad. Sci. USA 84:3344-3348 (1987); Yee et al., Cold Spring Harbor Symp.on Quant. Biol. Vol. LI, pp. 1021-1026 (1986); Wolff et al., Proc. Natl.Acad. Sci. U.S.A. 84:3344-3348 (1987); Jolly et al., Meth. in Enzymol.149:10-25 (1987); Miller et al., Mol. Cell. Biol. 5:431-437 (1985); andMiller, et al., Mol. Cell. Biol. 6:2895-2902 (1986) and Eglitis et al.,Biotechniques 6:608-614 (1988).

The effector and reporter molecules of the invention can be introducedinto herpes viruses (e.g. HSV-1). Herpes viruses are capable ofestablishing a latent infection and an apparently non-pathogenicrelationship with some neural cells.

Other virus vectors that may be used for gene transfer into cells fortreatment of brain tumors include retroviruses such as Moloney murineleukemia virus (MoMuLV); papovaviruses such as JC, SV40, polyoma,adenoviruses; Epstein-Barr Virus (EBV); papilloma viruses, e.g. bovinepapilloma virus type I (BPV); paramyxoviruses; vaccinia; rabies andpoliovirus and other human and animal viruses.

The nucleic acid molecules can be introduced into a cell or subjectusing retroviral vectors, or infectious viral particles, ornon-infectious particles (e.g., helper-dependent). The virus vector canbe modified so that it maintains the necessary genes, regulatorysequences and packaging signals to synthesize new viral proteins and RNAbut lacks genes conferring oncogenic potential.

In the case of non-infectious viral vectors, a helper virus genome isrequired to provide the structural genes necessary to encode viralstructural proteins. The helper virus lacks the viral packaging signalrequired to encapsulate the helper viral RNA into viral particles. Thus,only the helper-dependent viral vector carrying the heterologous genesequence and a functional packaging signal, but lacking viral structuralgenes can be incorporated into a virus particle. The helper andhelper-dependent vectors are used to infect a target cell and produce nofurther infectious virus can be produced since there are no viralstructural genes provided. Methods for constructing and using viralvectors are well known in the art and reviewed, for example, in Millerand Rosman, Biotechniques 7:980-990 (1992) and Davison and Elliot,Molecular Virology: A Practical Approach (IRL Press, New York, 1993).

The viral or other vector can include a constitutive promoter forconstitutive transcription of the transactivator sequence. Examples ofconstitutive promoters include the cytomegalovirus promoter (Boshart, M.et al., 1985 Cell 41:521-530). Viral vectors can include tissue-specificor tumor specific transcription or regulatory sequences to regulate thetype of cell that expresses the heterologous gene product byincorporating a tissue-specific promoter or enhancer into the vector(Dai et al., Proc. Natl. Acad. Sci. USA 89:10892-10895 (1992).

The preferred vectors permit transcription and translation of theeffector and/or reporter sequences in prokaryotic or eukaryotic hostcells.

The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate to the cell system in use (Scharf, D., et al, 1994Results Probl. Cell. Differ. 20:125-62; Bittner, et al., 1987 Methods inEnzymol. 153:516-544). The enhancers, transcriptional elements, andinitiation codons can be of various origins, both natural and synthetic.

The vectors having the effector or reporter sequences include expressionvectors which are compatible with prokaryotic host cells. Prokaryoticcell expression vectors are well known in the art and are available fromseveral commercial sources. For example, pET vectors (e.g., pET-21,Novagen Corp.), BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.),pSPORT (Gibco BRL, Rockville, Md.), or ptrp-lac hybrids for expressionin bacterial host cells.

Alternatively, the vectors having the effector or reporter sequences areexpression vectors which are compatible with eukaryotic host cells. Themore preferred vectors are those compatible with vertebrate cells.Eukaryotic cell expression vectors are well known in the art and areavailable from several commercial sources. Typically, such vectors areprovided containing convenient restriction sites for insertion of thedesired DNA segment. Typical of such vectors are PSVL and pKSV-10(Pharmacia), pBPV-1/pML2d (International Biotechnologies, Inc.), pTDT1(ATCC, #31255), and similar eukaryotic expression vectors.

Methods for generating a recombinant vector including the effector orreporter sequences are well known in the art, and can be found inManiatis, T., et al., (1989 in: “Molecular Cloning, A LaboratoryManual”, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) andAusubel et al. (1989 in: “Current Protocols in Molecular Biology”, JohnWiley & Sons, New York N.Y.).

The vector can include at least one selectable marker gene that encodesa gene product that confers drug resistance such as resistance tokanamycin, neomycin, tetracycline, or ampicillin. The selectable markerscan include dihydrofolate reductase or glutamine synthetase. The vectoralso comprises multiple endonuclease restriction sites that enableconvenient insertion of exogenous DNA sequences.

Host-Vector Systems

The invention further provides a host-vector system comprising a vector,plasmid, phagemid, or cosmid comprising the effector or reporternucleotide sequence, or a fragment thereof, introduced into a suitablehost cell. In one embodiment, the host cell is introduced with a vectorcomprising both the effector and reporter nucleotide sequences of theinvention. In another embodiment, the host cell is introduced with twoor more vectors (preferably two) where one vector comprises the effectornucleotide sequences of the invention and another vector comprises thereporter nucleotide sequences of the invention.

A variety of expression vector/host systems may be utilized to carry andexpress the effector or reporter sequences. The host-vector system canbe used to transcribe and translate express (e.g., produce) the effectortransactivator protein or the reporter heterologous gene product. Thehost cell can be either prokaryotic or eukaryotic. Examples of suitableprokaryotic host cells include bacteria strains from genera such asEscherichia, Bacillus, Pseudomonas, Streptococcus, and Streptomyces.Examples of suitable eukaryotic host cells include yeast cells, plantcells, or animal cells such as mammalian cells. A preferred embodimentprovides a host-vector system comprising the pBCVP2G5-L vector (Example2) in mammalian cells. A most preferred embodiment provides ahost-vector system comprising the pBCVP2G5-L vector (Example 2) in humanprostate cancer cells.

Introduction of the recombinant DNA molecules or vectors of the presentinvention into an appropriate host cell is accomplished by well knownmethods that depend on the type of vector used and host system employed.For example, host cells are introduced (e.g., transformed) with nucleicacid molecules by electroporation or salt treatment methods, see forexample, Cohen et al., 1972 Proc Acad Sci USA 69:2110; Maniatis, T., etal., 1989 in: “Molecular Cloning, A Laboratory Manual”, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. Host cells can also beintroduced with nucleic acid molecules by various methods, includingliposomes, cationic lipid, salt treatment (Graham et al., 1973 Virol52:456; Wigler et al., 1979 Proc Natl Acad Sci USA 76:1373-76),diethyaminoethyl dextran, or viral infection. Alternatively, particlebombardment can be used to introduce the vectors of the invention into ahost cell (Williams et al., Proc. Natl. Acad. Sci. USA 88:2726-2730(1991).

Successfully transformed cells, i.e., cells that harbor the vector ofthe present invention, can be identified by techniques well known in theart. For example, cells resulting from the introduction of the vectorare selected and cloned to produce single colonies. Cells from thosecolonies are harvested, lysed and their DNA content examined for thepresence of the rDNA using a method such as that described by Southern,J Mol Biol (1975) 98:503, or Berent et al., Biotech (1985) 3:208, or theproteins produced from the cell assayed via a biochemical assay orimmunological method.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the effector or reporter nucleotidesequences. The vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), pIN vectors (Van Heeke & Schuster (1989) J BiolChem 264:5503-5509), and the like. The pGEX vectors (Promega; Madison,Wis.) may also be used to express fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems are designed to includeheparin, thrombin or factor XA protease cleavage sites so that thecloned protein of interest can be released from the GST moiety at will.

In yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as beta-factor, alcohol oxidaseand PGH may be used. For reviews, see Ausubel et al (1989 in: “CurrentProtocols in Molecular Biology”, John Wiley & Sons, New York N.Y.) andGrant et al (1987) Methods in Enzymology 153:516-544.

In cases where plant expression vectors are used, the vectors having theeffector or reporter sequences of the invention can include viralpromoters such as the 35S and 19S promoters of CaMV (Brisson, et al.,(1984) Nature 310:511-514), or the omega leader sequence from TMV(Takamatsu, et al., (1987) EMBO J. 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO (Coruzzi et al (1984)EMBO J. 3:1671-1680; Broglie et al (1984) Science 224:838-843); or heatshock promoters (Winter J and Sinibaldi R M (1991) Results Probl CellDiffer 17:85-105) can be used. These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. For reviews of such techniques, see Hobbs, S. in: “McGrawYearbook of Science and Technology” 1992 McGraw Hill New York N.Y., pp191-196; or Weissbach and Weissbach 1988 in: “Methods for PlantMolecular Biology”, Academic Press, New York N.Y., pp 421-463.

An alternative expression system that can be used to express theeffector and reporter sequences is an insect system. In one such system,Autographa californica nuclear polyhedrosis virus (AcNPV) can be used asa vector to express the effector transactivator and reporterheterologous gene product in Spodoptera frugiperda cells or inTrichoplusia larvae. The effector or reporter sequence can be clonedinto a nonessential region of the virus, such as the polyhedrin gene,and placed under control of the polyhedrin promoter. Successfulinsertion of the effector or reporter sequence will render thepolyhedrin gene inactive and produce recombinant virus lacking coatprotein. The recombinant viruses can then used to infect S. frugiperdacells or Trichoplusia larvae in which the effector transactivator or thereporter heterologous gene product can be expressed (Smith et al 1983 JVirol 46:584; Engelhard E. K., et al, 1994 Proc Nat Acad Sci 91:3224-7).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the effector or reporter sequence can be ligated into anadenovirus transcription/translation vector consisting of the latepromoter and tripartite leader sequence. Insertion in a nonessential E1or E3 region of the viral genome results in a viable virus capable ofexpressing the effector transactivator or the reporter heterologous geneproduct in infected host cells (Logan and Shenk 1984 Proc Natl Acad Sci81:3655-59). In addition, transcription enhancers, such as the roussarcoma virus (RSV) enhancer, can be used to increase expression inmammalian host cells.

The host cell may be isolated from various animal sources includingequine, porcine, bovine, murine, canine, feline, or avian. The host cellmay also be isolated from any mammalian source including monkey, ape, orhuman. The host cell may be isolated from various cell or organ sourcesincluding blood, prostate, brain, lung, stomach, bladder, pancreas,colon, breast, ovary, uterus, cervix, liver, muscle, skin, or bone.

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of theprotein include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing which cleaves a precursor form of aprotein (e.g., a prepro protein) may also be important for correctinsertion, folding and/or function. Different host cells such as CHO,HeLa, MDCK, 293, WI38, etc. have specific cellular machinery andcharacteristic mechanisms for such post-translational activities and maybe chosen to ensure the correct modification and processing of theeffector or reporter gene products.

For long-term, high-yield production of the effector transactivator orthe reporter heterologous gene product, stable expression is preferred.For example, cell lines that stably express the effector or the reportergene product can be transformed using expression vectors that containviral origins of replication or endogenous expression elements and aselectable marker gene. Following the introduction of the vector, cellscan be grown in an enriched media before they are switched to selectivemedia. The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clumps ofstably transformed cells can be proliferated using tissue culturetechniques appropriate for the cell type used.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M., et al., 1977 Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al., 1980 Cell 22:817-23) whichcan be employed in tk-minus or aprt-minus cells, respectively. Also,anti-metabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M., et al., 1980 Proc Natl Acad Sci 77:3567-70);npt, which confers resistance to the aminoglycosides neomycin and G-418(Colbere-Garapin, F., et al., 1981 J. Mol. Biol. 150:1-14) and als orpat, which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively. Additional selectable genes have beendescribed, for example, trpB, which allows cells to utilize indole inplace of tryptophan, or hisD, which allows cells to utilize histinol inplace of histidine (Hartman, S. C. and R. C. Mulligan 1988 Proc. Natl.Acad. Sci. 85:8047-51). Recently, the use of visible markers has gainedpopularity with such markers as anthocyanins, β-glucuronidase and itssubstrate, GUS, and luciferase and its substrate, luciferin, beingwidely used not only to identify transformants, but also to quantify theamount of transient or stable protein expression attributable to aspecific vector system (Rhodes, C. A., et al., 1995 Methods Mol. Biol.55:121-131).

Nucleic Acid Molecules

In its various aspects the present invention provides recombinantnucleic acid molecules, transformed host cells harboring the nucleicacid molecules, generation methods, and assays.

The nucleic acid molecules of the invention are preferably in isolatedform, where the nucleic acid molecules are substantially separated fromcontaminant nucleic acid molecules having sequences other than theeffector or reporter sequences. A skilled artisan can readily employnucleic acid isolation procedures to obtain isolated effector orreporter sequences, see for example Sambrook et al., in: “MolecularCloning” (1989). The present invention also provides for isolatedeffector or reporter sequences generated by recombinant DNA technologyor chemical synthesis methods.

The isolated nucleic acid molecules include DNA, RNA, DNA/RNA hybrids,and related molecules, nucleic acid molecules complementary to theeffector or reporter sequences or a portion thereof, and those whichhybridize to the effector or reporter sequences. The preferred nucleicacid molecules have nucleotide sequences identical to or nearlyidentical (e.g., similar) to the nucleotide sequences disclosed herein.Specifically contemplated are DNA, cDNA, RNA, ribozymes, and antisensemolecules.

Identical and Variant Sequences

The present invention provides isolated nucleic acid molecules having apolynucleotide sequence identical or similar to the effector or reportersequences disclosed herein. Accordingly, the polynucleotide sequencesmay be identical to a particular effector or reporter sequence.Alternatively, the polynucleotide sequences may be similar to thedisclosed sequences.

One embodiment of the invention provides nucleic acid molecules thatexhibit sequence identity or similarity with the effector or reporternucleotide sequences, such as molecules that have at least 60% to 99.9%sequence similarity and up to 100% sequence identity with the sequencesof the invention. A preferred embodiment provides nucleic acid moleculesthat exhibit between about 75% to 99.9% sequence similarity, a morepreferred embodiment provides molecules that have between about 86% to99.9% sequence similarity, and the most preferred embodiment providesmolecules that have 100% sequence identity with the effector or reportersequences of the invention.

Complementary Sequences

The invention also provides nucleic acid molecules that arecomplementary to the effector or reporter sequences of the invention.Complementarity may be full or partial. When it is fully complementarythat means complementarity to the entire sequence. When it is partiallycomplementary that means complementarity to only portions of thesequences of the invention.

Nucleotide Sequences which Hybridize

The present invention further provides nucleotide sequences thatselectively hybridize to the effector or reporter nucleotide sequencesof the invention under high stringency hybridization conditions.Typically, hybridization under standard high stringency conditions willoccur between two complementary nucleic acid molecules that differ insequence complementarity by about 70% to about 100%. It is readilyapparent to one skilled in the art that the high stringencyhybridization between nucleic acid molecules depends upon, for example,the degree of identity, the stringency of hybridization, and the lengthof hybridizing strands. The methods and formulas for conducting highstringency hybridizations are well known in the art, and can be foundin, for example, Sambrook, et al., in: “Molecular Cloning” (1989).

In general, stringent hybridization conditions are those that: (1)employ low ionic strength and high temperature for washing, for example,0.015M NaCl/0.0015M sodium titrate/0.1% SDS at 50 degrees C.; or (2)employ during hybridization a denaturing agent such as formamide, forexample, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM NaCl, 75 mM sodium citrate at 42 degrees C.

Another example of stringent conditions include the use of 50%formamide, 5×SSC (0.75M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution,sonicated salmon sperm DNA (50 mg/ml), 0.1% SDS, and 10% dextran sulfateat 42 degrees C., with washes at 42 degrees C. in 0.2×SSC and 0.1% SDS.A skilled artisan can readily determine and vary the stringencyconditions appropriately to obtain a clear and detectable hybridizationsignal.

Fragments

The invention further provides nucleic acid molecules having fragmentsof the effector or reporter sequences of the invention. The size of thefragment will be determined by its intended use. For example, if thefragment is chosen to be used as a nucleic acid probe or PCR primer,then the fragment length is chosen to obtain a relatively small numberof false positives during a probing or priming procedure.

The nucleic acid molecules, fragments thereof, and probes and primers ofthe present invention are useful for a variety of molecular biologytechniques including, for example, hybridization screens of libraries,or detection and quantification of mRNA transcripts as a means foranalysis of gene transcription and/or expression. The probes and primerscan be DNA, RNA, or derivative molecules including peptide nucleic acids(PNAs). A probe or primer length of at least 15 base pairs is suggestedby theoretical and practical considerations (Wallace, B. and Miyada, G.1987 in: “Oligonucleotide Probes for the Screening of Recombinant DNALibraries” in: “Methods in Enzymology”, 152:432-442, Academic Press).

Fragments of the effector or reporter sequences that are particularlyuseful as selective hybridization probes or PCR primers can be readilyidentified from the effector or reporter nucleotide sequences, usingart-known methods. For example, sets of PCR primers that detect aportion of the effector or reporter transcripts can be made by the PCRmethod described in U.S. Pat. No. 4,965,188. The probes and primers ofthis invention can be prepared by methods well known to those skilled inthe art (Sambrook, et al. supra). In a preferred embodiment the probesand primers are synthesized by chemical synthesis methods (ed: Gait, M.J. 1984 in: “Oligonucleotide Synthesis”, IRL Press, Oxford, England).

One embodiment of the present invention provides nucleic acid primersthat are complementary to effector or reporter sequences, which allowthe specific amplification of nucleic acid molecules of the invention orof any specific portions thereof. Another embodiment provides nucleicacid probes that are complementary for selectively or specificallyhybridizing to the effector or reporter sequences or to any portionthereof.

Codon Usage Variants

The present invention provides isolated codon-usage variants that differfrom the disclosed effector or reporter nucleotide sequences, yet do notalter the predicted polypeptide sequence or biological activity of theeffector gene product (e.g, the transactivator protein) or the reportergene product (e.g., the heterologous gene product). For example, anumber of amino acids are encoded by more than one codon triplet. Codonsthat specify the same amino acid, or synonyms may occur due todegeneracy in the genetic code. Examples include nucleotide codons CGT,CGG, CGC, and CGA encoding the amino acid, arginine (R); or codons GAT,and GAC encoding the amino acid, aspartic acid (D). Thus, a protein canbe encoded by one or more nucleic acid molecules that differ in theirspecific nucleotide sequence, but still encode protein molecules havingidentical sequences. The amino acid coding sequence is as follows: OneAmino Acid Symbol Symbol Letter Codons Alanine Ala A GCU, GCC, GCA, GCGCysteine Cys C UGU, UGC Aspartic Acid Asp D GAU, GAC Glutamic Acid Glu EGAA, GAG Phenylalanine Phe F UUU, UUC Glycine Gly G GGU, GGC, GGA, GGGHistidine His H CAU, CAC Isoleucine Ile I AUU, AUC, AUA Lysine Lys KAAA, AAG Leucine Leu L UUA, UUG, CUU, CUC, CUA, CUG Methionine Met M AUGAsparagine Asn N AAU, AAC Proline Pro P CCU, CCC, CCA, CCG Glutamine GlnQ CAA, CAG Arginine Arg R CGU, CGC, CGA, CGG, AGA, AGG Serine Ser S UCU,UCC, UCA, UCG, AGU, AGC Threonine Thr T ACU, ACC, ACA, ACG Valine Val VGUU, GUC, GUA, GUG Tryptophan Trp W UGG Tyrosine Tyr Y UAU, UAC

The codon-usage variants may be generated by recombinant DNA technology.Codons may be selected to optimize the level of production of theeffector or reporter transcript, or the effector or reporter geneproduct in a particular prokaryotic or eukaryotic expression host, inaccordance with the frequency of codon utilized by the host cell.Alternative reasons for altering the nucleotide sequence encoding aneffector or reporter transcript include the production of RNAtranscripts having more desirable properties, such as an extendedhalf-life or increased stability. A multitude of variant effector orreporter nucleotide sequences that encode the respective effector orreporter gene products may be isolated, as a result of the degeneracy ofthe genetic code. Accordingly, the present invention provides selectingevery possible triplet codon to generate every possible combination ofnucleotide sequences that encode the disclosed effector or reporter geneproducts, or that encode the gene products having the biologicalactivity of the effector or reporter gene products. This particularembodiment provides isolated nucleotide sequences that vary from thesequences of the invention, such that each variant nucleotide sequenceencodes a polypeptide having sequence identity with the amino acidsequences of the invention.

Derivative Nucleic Acid Molecules

The nucleic acid molecules of the invention also include derivativenucleic acid molecules which differ from DNA or RNA molecules, andanti-sense molecules. Derivative molecules include peptide nucleic acids(PNAs), and non-nucleic acid molecules including phosphorothioate,phosphotriester, phosphoramidate, and methylphosphonate molecules, thatbind to single-stranded DNA or RNA in a base pair-dependent manner(Zamecnik, P. C., et al., 1978 Proc. Natl. Acad. Sci. 75:280284;Goodchild, P. C., et al., 1986 Proc. Natl. Acad. Sci. 83:4143-4146).Peptide nucleic acid molecules comprise a nucleic acid oligomer to whichan amino acid residue, such as lysine, and an amino group have beenadded. These small molecules, also designated anti-gene agents, stoptranscript elongation by binding to their complementary (template)strand of nucleic acid (Nielsen, P. E., et al., 1993 Anticancer Drug Des8:53-63). Reviews of methods for synthesis of DNA, RNA, and theiranalogues can be found in: “Oligonucleotides and Analogues”, eds. F.Eckstein, 1991, IRL Press, New York; “Oligonucleotide Synthesis”, ed. M.J. Gait, 1984, IRL Press, Oxford, England. Additionally, methods forantisense RNA technology are described in U.S. Pat. Nos. 5,194,428 and5,110,802. A skilled artisan can readily obtain these classes of nucleicacid molecules using the herein described effector and reportersequences (see for example “Innovative and Perspectives in Solid PhaseSynthesis” (1992) Egholm, et al. pp 325-328 or U.S. Pat. No. 5,539,082).

RNA Molecules

The present invention provides nucleic acid molecules that encode theeffector or reporter gene products. In particular, the RNA molecules ofthe invention may be isolated full-length or partial-length RNAmolecules or RNA oligomers that encode the effector or reporter geneproducts.

The RNA molecules of the invention also include antisense RNA molecules,peptide nucleic acids (PNAs), or non-nucleic acid molecules such asphosphorothioate derivatives, that specifically bind in a base-dependentmanner to the sense strand of DNA or RNA, having the effector orreporter sequences, in a base-pair manner. A skilled artisan can readilyobtain these classes of nucleic acid molecules using the effector orreporter sequences described herein.

Labeled Nucleic Acid Molecules

Embodiments of the effector or reporter nucleic acid molecules of theinvention include DNA and RNA primers, which allow the specificamplification of the effector or reporter sequences, or of any specificparts thereof, and probes that selectively or specifically hybridize tothe effector or reporter sequences or to any part thereof. The nucleicacid probes can be labeled with a detectable marker. Examples of adetectable marker include, but are not limited to, a radioisotope, afluorescent compound, a bioluminescent compound, a chemiluminescentcompound, a metal chelator or an enzyme. Technologies for generatinglabeled DNA and RNA probes are well known, see, for example, Sambrook etal., in “Molecular Cloning” (1989).

Methods for Producing the Heterologous Gene Product

The present invention provides methods for producing the heterologousgene product. These methods utilize a cell introduced with the effectorand reporter nucleic acid molecules of the invention. The cell can beintroduced with nucleic acid molecules of the invention inserted into avector that permits transcription of the effector transactivator and theheterologous gene sequence. The cell, harboring the nucleic acidmolecules of the invention, is induced to produce the heterologous geneproduct. Alternatively, the cell produces the heterologous gene productconstitutively. The heterologous gene product, produced by thesemethods, can be detected and/or imaged in the cell or in the subject.

In one embodiment, the steps include: introducing a cell with thenucleic acid molecules of the invention; and culturing the introducedcell under conditions suitable for production of the heterologous geneproduct. The conditions which are suitable for production of theheterologous gene product include contacting the cell with an agent thatinduces transcription of the effector transactivator. The transcript istranslated into the transactivator protein which activates transcriptionof the reporter nucleic acid molecule and translation of theheterologous gene product.

The agent includes agents that are capable of causing transcription ofthe transcription regulatory sequences in the effector molecule. Forexample, the inducing agent can be an androgen that mediatestranscription of the effector molecule having a prostate-specific orprostate tumor-specific promoter and/or enhancer. The inducing androgencan be natural or synthetic.

In another embodiment, the methods for producing the heterologous geneproduct include: introducing the nucleic acid molecules of the inventioninto a subject under conditions that permit production of theheterologous gene product.

The nucleic acid molecules are introduced into the subject via variousroutes, including intravenous, intraperitoneal, intramuscular,intratumoral, intradermal, subcutaneous, and the like. The nucleic acidmolecules of the invention can be introduced at a site distal from atarget site, such as a tumor. The heterologous gene product produced bythe nucleic acid molecules of the invention can migrate to the targetsite. For example, the nucleic acid molecules of the invention can beintroduced into the subject intramuscularly and the heterologous geneproduct can migrate to the target tumor via the circulatory system.Alternatively, the nucleic acid molecules of the invention can beintroduced at the target site, such as in or proximal to the tumor.

The conditions which are suitable for production of the heterologousgene product include contacting the cell, so introduced, with an agentthat induces transcription of the effector transactivator. Thetranscript is translated into the transactivator protein which activatestranscription of the reporter nucleic acid molecule and translation ofthe heterologous gene product.

The present invention also provides methods for producing theheterologous gene product which include: implanting a donor cell into asubject under conditions that permit production of the heterologous geneproduct, where the donor cell harbors the nucleic acid molecules of theinvention. The conditions which are suitable for production of theheterologous gene product include contacting the donor cell, soimplanted, with an agent that induces transcription of the effectortransactivator. The transcript is translated into the transactivatorprotein which activates transcription of the reporter nucleic acidmolecule and translation of the heterologous gene product.

The donor cells can be introduced into the subject as a cell suspensionor as a solid clump of cells. The donor cells can be introduced into thesubject via various routes, including intravenous, intraperitoneal,intramuscular, intratumor, intradermal, subcutaneous, and the like. Thedonor cells can be implanted at a site distal to the target site, suchas a tumor. In such cases, the heterologous gene product is produced bythe donor cell and can migrate to the target site. For example, thedonor cell can be implanted into the subject intramuscularly and theheterologous gene product can migrate to a prostate tumor target sitevia the circulatory system. Alternatively, the donor cells can beimplanted in or proximal to the target site.

The donor cell can be a cell from a the subject (e.g., autologous cells)or from a different subject (e.g., non-autologous). Donor cells whichharbor the molecules of the invention include any cell from a subject,including blood, prostate, brain, lung, stomach, bladder, pancreas,colon, breast, ovary, uterus, cervix, liver, muscle, skin, or bone.Cells which harbor the molecules of the invention include any cell fromany animal including mouse, rat, rabbit, dog, cat, horse, cow, goat,pig, fish, monkey, ape or human. Non-autologous cells such as allogeneiccells also can be used provided they share histocompatibility antigenswith the subject to be introduced.

Methods for preparing donor cells and implanting donor cells harboringrecombinant retroviral vectors carrying a heterologous gene sequencehave been described (Gage et al., Ch. 86, in: “Progress in BrainResearch”, Vol. 78, pp. 651-658, 1988; U.S. Pat. No. 5,529,774).

A variety of methods can be used to introduce the nucleic acid moleculesof the invention into a cell, into a subject, or into a donor cell whichis implanted into a subject.

The molecules of the invention can be introduced as naked nucleic acidmolecules (with or without a linked vector), liposomes, virus vectors,viruses, or receptor-mediated agents.

In one embodiment, introduction of naked nucleic acid molecules orliposomes (e.g, cationic) can be used for stable gene transfer of thenucleic acid molecules. The nucleic acid molecules can be introducedinto non-dividing or dividing cells in vivo (Ulmer et al., Science259:1745-1748 (1993). In addition, the nucleic acid can be transferredinto a variety of tissues in vivo using the particle bombardment method(Williams et al., Proc. Natl. Acad. Sci. USA 88:2726-2730 (1991).

The nucleic acid molecules can be encapsulated into liposomes usinghemagglutinating viruses including the Japan Sendai virus (Z strain). Inone embodiment, the nucleic acid molecules are linked to a vectorencapsulated into a liposome HVJ construct prior to injection.Alternatively, the liposomes can be produced using Lipofectamine™. Thenucleic acid molecules can be introduced into a cell or a subject viaviral vectors or viruses. The selection of a viral vector will depend,in part, on the cell type to be targeted. Specialized viral vectors arewell known in the art that can target specific cell types.

The molecules of the invention can be introduced into a cell or asubject using receptor-mediated delivery methods. The nucleic acidmolecules of the invention can be complexed with a tissue-specificligand. The ligand can be an agent that binds a cell receptor.Alternatively, the ligand can be an antibody that binds a cell receptor.The nucleic acid molecules of the invention can be complexed with theligand by a covalent, or non-covalent link such as a bridging molecule(Curiel et al., Hum. Gene Ther. 3:147-154 (1992); Wu and Wu, J. Biol.Chem. 262:4429-4432 (1987).

Methods for Detecting Production of the Heterologous Gene Product

The present invention provides methods for detecting the production ofthe heterologous gene product. The methods of the invention compriseintroducing the effector and reporter nucleic acid molecules of theinvention into a cell (e.g., transfected cell) under conditions suitablefor production of the heterologous gene product; and detection of theheterologous gene product. The detecting methods of the invention areperformed using a cultured transfected cell, a cell transfected in asubject, or a donor cell implanted in a subject.

The heterologous gene product, which is produced by these transfectedcells, is detected using non-invasive radionuclide and/ornon-radionuclide techniques that permit detection of the location,magnitude and time variation of the heterologous gene product (P. Ray,et al., 2001 Semin. Nucl. Med. 31:312-320).

The detecting step includes non-invasive techniques such as positronemission tomography (e.g., PET) (S. Gambhir, et al., 1999 Proc. Natl.Acad. Sci. USA 96:2333-2338; J G Tjuvajev, et al., 1998 Cancer Res58:4333-4341; S R Cherry, et al., 1997 IEEE Transactions on NuclearScience 44:1161-1166; M E Phelps, 1991 Neurochemical Research16:929-994; S R Cherry and S S Gambhir 2001 Inst. Lab. Anim. Res. J.42:219-232), single photon emission computed tomography (J G. Tjuvajev,et al., 1996 Cancer Res. 45:4087-4095; K R Zinn, et al., 2000 J. Nucl.Med. 41:887-895), cooled charged coupled device (CCD) camera opticalimaging (Honigman, et al., 2001 Mol. Ther. 4:239-249); magneticresonance imaging (A H Louie, et al., 2000 Nat. Biotechnol. 18:321-325;R. Weissleder, et al., 2000 Nat. Med. 6:351-355), bioluminescent opticalimaging (P R Contag, et al., 1998 Nat. Med. 4:245-247; A Honigman, etal., 2001 Mol. Ther. 4:239-249; J C Wu, et al., 2001 Mol. Ther.4:297-306; M Iyer, et al., 2001 Proc. Natl. Acad. Sci. USA98:14595-14600), and fluorescence optical imaging (M Yang, et al., 2001Proc. Natl. Acad. Sci USA 98:2616-2621).

The type of non-invasive technique used to detect the heterologous geneproduct is selected based on the type of gene product produced. Forexample, bioluminescent optical imaging (P R Contag, et al., 1998 Nat.Med. 4:245-247; J C Wu, et al., 2001 Mol. Ther. 4:297-306) has been usedto detect production of luciferase.

Alternatively, cooled charge-coupled device camera has been used todetect luciferase in whole animals (A Honigman, et al., 2001 Mol. Ther.4:239-249; M Iyer, et al., 2001 Proc. Natl. Acad. Sci. USA98:14595-14600).

In other cases, positron emission tomography or micro PET andradiolabeled tracers have been used to detect a thymidine kinase or amutant version of thymidine kinase, HSV-sr39tk (S R Cherry, et al., 1997IEEE Transactions on Nuclear Science 44:1161-1166; S. Gambhir, et al.,1999 Proc. Natl. Acad. Sci. USA 96:2333-2338; S S Gambhir, et al., 1998J. Nucl. Med. 39:2003-2011; D C MacLaren, et al., 1999 Gene Ther.6:785-79; M Iyer, et al., 2001 J. Nucl. Med. 42:96-105; S S Gambhir, etal., 2000 Proc. Natl. Acad. Sci. USA 97:2785-2790; S S Gambhir, et al.,1999 Journal of Nuclear Cardiology 6:219-233).

Positron emission tomography and radiolabeled spiperone({(3-(2′[¹⁸F]-fluorethyl)spiperone (FESP)}has also been used to detectdopamine type 2 receptor (D₂R) (D C MacLaren, et al., 1998 Journal ofNuclear Medicine 39:35P; D C MacLaren, et al., 1999 Gene Therapy6:785-791).

In one case, production of thymidine kinase and dopamine type 2 receptorhave been simultaneously detected using microPET (M. Iyer, et al., 2001Journal of Nuclear Medicine 42:96-105).

Magnetic resonance spectroscopy has been used to detect production ofthe pro-drug enzyme cytosine deaminase (L D Stegman, et al., 1999 Proc.Natl. Acad. Sci. USA 96:9821-9826; U Habrerkorn, et al., 1996 Journal ofNuclear Medicine 37:87-94).

The production of somatostatin receptor subtype II (SSTr2) has beendetected using radiolabeled octreotide (B E Rogers, et al., 2000Quarterly Journal of Nuclear Medicine 44:208-223).

The following examples are intended to illustrate, but not limit, thescope of the invention.

EXAMPLE 1

The following example provides description of a two-step transcriptionalamplification as a method for imaging reporter gene expression usingweak promoters.

Assays for imaging reporter gene expression are very useful forvisualizing molecular events in living animals. Non-invasive imaging ofreporter gene expression by using radionuclide and non-radionuclidetechniques allows the monitoring of the location, magnitude, and timevariation of gene expression in living animals and, in some cases,humans. Applications of reporter genes include: (i) optimization of genetherapy vectors that contain a reporter gene, (ii) imaging celltrafficking by marking cells with a reporter gene ex vivo, and (iii)monitoring endogenous gene expression through the use of reporter genescoupled to endogenous promoters in transgenic models. All of theseapplications should benefit from repetitive, noninvasive monitoring ofreporter gene expression.

Methods to image reporter gene expression in living animals includepositron emission tomography (PET) (1, 2), single photon emissioncomputed tomography (3, 4), magnetic resonance imaging (5, 6),bioluminescent optical imaging with firefly luciferase (fl) (7-9), andfluorescence optical imaging with green fluorescent protein (10). Adetailed review of the different approaches for imaging reporter genesis reported elsewhere (11).

The cooled charge-coupled device (CCD) camera offers a convenient,cost-effective, and reproducible method to image fl expression in smallliving animals (7-9). Recently, real-time monitoring of fl reporter geneexpression in various tissues has been evaluated in rodents by using acooled CCD camera (8). In this article, the authors have demonstratedthat injection of adenovirus, AdHIV/fl (the fl expression is driven bythe HIV-long-terminal repeat promoter) results in fl expression in theregion over the testis in male mice. Transgenic mice with the human boneγ-carboxyglutamate protein promoter driving fl led to gene expression inbones and teeth. The use of tissue-specific promoters in these limitedapplications was possible, but in many applications specific promotersmay not lead to sufficient levels of fl expression. In other cases,enhancement of observable signal may benefit from amplification.

The advantage of PET over optical approaches is the ability to obtaintomographic and quantitative information with high sensitivity. Withappropriate corrections for photon attenuation, scatter, and objectsize, concentrations of radiolabeled tracers can be reliably estimated(12). Furthermore, unlike optical methods, small animal imaging withmicroPET (13) directly translates to human studies with clinical PETscanners. We have previously developed methods to monitoradenoviral-mediated herpes simplex virus type 1 thymidine kinase(HSV1-tk) expression in vivo by using 18 F-labeled acycloguanosineanalogs and microPET (1, 14). We have studied cell lines stablytransfected ex vivo with various PET reporter genes and implanted inmice for imaging with microPET (15, 16). We also have validated the useof a mutant HSV1-sr39tk with enhanced imaging sensitivity (17). In ourearlier approaches, the expression of the PET reporter gene was drivenby relatively strong promoters [e.g., cytomegalovirus (CMV)] thatachieve a constitutive, high level of transcription. In many therapeuticapplications, targeted approaches to enhance safety and specificity arehighly desirable. To this end, transcriptional targeting by usingtissue-specific promoters to limit expression of potential cytotoxictransgenes to the tissue of interest has been frequently used (18-20).In applications where a relatively weak, cellular promoter drives theexpression of a reporter gene, one has to contend with weakertranscriptional activity that, in turn, limits the ability to imagereporter gene expression in vivo. In such cases, it is essential to findways to enhance the transcriptional activity of such promoters. Severalpotential methods can be used to increase levels of reporter protein.These include (i) increased transcription by using chimeric promotersthat retain tissue specificity (20, 21), (ii) enhancement at theposttranscriptional level (22-24), or (iii) transcriptionalamplification approaches discussed next.

One of the amplification approaches referred to as a two-steptranscriptional amplification (activation) (TSTA) approach that canpotentially be used to augment the transcriptional activity of cellularpromoters uses the GAL4-VP16 fusion protein (25, 26) (FIG. 1). Thisapproach is also referred to as a recombinant transcriptional activationapproach (27). The yeast GAL4 gene expression system is one of the mostwidely studied eukaryotic transcriptional regulatory systems (28).

We are presently evaluating methods to non-invasively image reportergene expression driven by cellular promoters. We chose to evaluate theprostate-specific antigen promoter (named PSE in this article) as it hasbeen well characterized, determined to be tissue-specific, and isexpressed after androgen administration in cell culture and in vivo(29). In this study, we chose to take advantage of the strongtransactivating properties of the GAL4-VP16 fusion protein to achieveprostate-specific amplification by using the PSE promoter to driveexpression of fl or HSV1-sr39tk in an LNCaP prostate cancer cell line.We also validated the system for monitoring reporter gene expression inliving mice implanted with LNCaP cells transiently expressing fl byusing a cooled CCD optical imaging system.

Materials and Methods

TSTA Experimental Strategy

The HSV1 VP16 immediate early transactivator contains a highly potentactivation domain that, when fused to the GAL4 DNA binding domain,elicits a robust response on a GAL4-responsive promoter (referred to asa minimal promoter in this work) bearing multiple tandem copies of the17 bp near consensus DNA binding site (25, 26). The VP16 activationdomain amino acids 411-490 can be subdivided further into N- andC-terminal subdomains. The C-terminal domain is sometimes toxic to cellsand elicits a transcriptional inhibitory phenomenon called squelching(30). GAL4-VP16 is a fusion of the N-terminal portion of the VP16activation domain from amino acids 413-454 and the GAL4 DNA bindingdomain amino acids 1-147. It is a potent activator that squelches lesseffectively than the intact activation domain.

Chemicals

Methyltrienolone (R1881) was purchased from NEN. Tfx-50 transfectionreagent and luciferase assay kit were purchased from Promega.D-Luciferin for use with in vivo fl imaging was obtained from Xenogen(Alameda, Calif.). [8-³H]Penciclovir (17.6 Ci/mmol) was purchased fromMoravek Biochemicals (Brea, Calif.). Testosterone pellets (0.007 mg, 6-hrelease) were obtained from Innovative Research of America. Matrigel waspurchased from BD Biosciences (Bedford, Mass.). Cell Culture. The humanprostate cancer cells, LNCaP (provided by C. Sawyers, University ofCalifornia, Los Angeles), were grown in RPMI 1640 supplemented with 10%FBS and 1% penicillin/streptomycin solution. The C6 rat glioma cellswere kindly provided by Margaret Black (Washington State University,Pullman) and were grown in deficient DMEM, supplemented with 5% FBS and1% penicillin/streptomycin/L-glutamine. HeLa cells (American TypeCulture Collection) were grown in DMEM with 10% FBS and 1%penicillin/streptomycin.

Construction of Plasmids/Vectors

The parental construct, PSE, derived from plasmid PSAR2.4k-PCPSA-P-Lux(29), consisted of a 2.4-kb enhancer fragment (−5322 to −2855) and theproximal promoter region from −541 to +12, upstream of an fl reportergene. PSE was chosen to be the baseline construct because the 2.4-kbenhancer fragment generates the maximal transcriptional andandrogen-responsive activity, comparable to the entire 6-kb regulatoryregion of the prostate-specific antigen gene (20, 31). The constructionof PSE plasmid has been described (20). To construct pBS-PSEGAL4VP16,the HindIII to XbaI GAL4VP16 fragment was excised frompSP72-SV40-GAL4VP16 and inserted into PSE plasmid. The minimal promoter,G5E4TATA (G5E4T) contained templates bearing five 17-bp GAL4 bindingsites positioned 23 bases from the TATA box of the E4 gene of adenovirus(32). The G5E4T-fl construct was made by PCR amplification of therelevant GAL4 sites and E4TATA from the pGEM3 G5E4T vector by using anupstream primer with a SacI site attached (cccgagctcatttaggtgacactatag)and a downstream primer with a XhoI site attached(cccctcgagacaccactcgacacggcacc). The PCR fragments were digested withSacI and XhoI and cloned into the pGL3 basic vector. The G5E4T-sr39tkplasmid was constructed by excising the G5E4T fragment frompSP72-G5E4T-CAT and cloning into pcDNA3.1 vector. The G5E4T sequence wasPCR-amplified by using primer pair 5′-gactagatctacagcttgcatgcctgcag-3′and 5′-gactgctagctcgacacg-gcacca-3′ and cloned into the BglII/NheI sitesupstream of sr39tk in the main vector, pcDNA3.1. For the sake ofconvenience, PSEGAL4-VP16 is abbreviated as PG, and the re-porterplasmid G5E4T-fl is abbreviated as L5. The reporter templateG5E4T-sr39tk is referred to as T5. The constructs PSE-fl,PSE-HSV1-sr39tk, and SV40-GAL4-VP16 are abbreviated as PL, PT, and SG,respectively.

Cell Transfections and Enzyme Assays

On day 1, LNCaP cells were plated in 6-well plates in RPMI 1640containing charcoal-stripped FBS. Transient transfections were performed24 h later by using Tfx-50 transfection reagent (Promega). Eachtransfection mix consisted of 0.5 micro grams of the effector andreporter plasmids or reporter plasmid alone. One hour aftertransfection, methyltrienolone (R1881) in ethanol was added to themedium at a concentration of 1 nM/well, and the cells were incubated for48 h. For FL activities (fl refers to the gene and FL to the enzyme),the cells were harvested and assayed for FL activity by using thedual-reporter luciferase assay system (Promega) and a luminometer (Lumat9507, Berthold, Germany) with an integration time of 10 s. LNCaP cellswere also transfected by using CMV-fl plasmid (as a positive control).For the TK assay, the cells were harvested 48 h after transfection andassayed for HSV1-sr39TK enzyme activity (tk refers to the gene and TK tothe enzyme) as described (14). For androgen inducibility experiments,LNCaP cells were transiently transfected with PG and T5 constructs inthe presence of different concentrations of androgen. After 48 h, thecells were assayed for HSV1-sr39TK activity.

In Vivo Optical Imaging of Fl Expression Using a Cooled CCD Camera

LNCaP cells were transiently transfected with the effector and reporterplasmids or reporter plasmid alone as described earlier. The cells wereharvested 48 h after transfection and resuspended in PBS. An aliquot of1×10⁶ cells was mixed with Matrigel and injected i.p. in female nudemice. Five minutes after injection of the cells, the mice wereanesthetized (ketaminexylazine, 4:1), and 200 μl of D-luciferin (15mg/ml) was injected (i.p.) 10 min before imaging. After the first scan,the mice were s.c. implanted with 0.007-mg sustained releasetestosterone pellets and imaged again after 24 h and 48 h. A total offive mice were used for the experiment, and one mouse in each group didnot receive the androgen pellet.

The mice were imaged by using a cooled CCD camera (Xenogen IVIS,Alameda, Calif.) with an acquisition time of 5 min. The animals wereplaced supine in a light-tight chamber, and a gray scale reference imagewas obtained under low-level illumination. Photons emitted from cellsimplanted in the mice were collected and integrated for a period of 5min. Images were obtained by using LIVING IMAGE software (Xenogen) andIGOR IMAGE analysis software (WaveMetrics, Lake Oswego, Oreg.). Forquantitation of measured light, regions of interest were drawn over theperitoneal region, and maximum relative light units (RLU) per min wereobtained as validated (9).

Results

TSTA System Mediates Prostate-Specific Amplification of Fl Expression inLNCaP Cells and Demonstrates Cell-Type Specificity

In transient transfection into LNCaP cells, when GAL4-VP16 was placedunder the control of the PSE promoter, it activated poorly in theabsence of androgen, but the response in the presence of androgen wassignificantly higher than that observed by using the reporter templatealone (L5) (FIG. 2A). The maximal level of fl expression is observed byusing five binding sites upstream of the adenovirus E4 promoter (L5) onthe reporter template vs. one or two binding sites. The TSTAsystem-driven amplification of fl expression in the presence of androgenis similar to the SG-activated expression of a GAL4-responsive flreporter in the absence and presence of androgen stimulation (−L/+L,FIG. 2A). These results validate the concept that the tissue-specific,ligand-responsive PSE promoter could be substituted for a strong viralone to elicit similar levels of GAL4-VP16 functional activity. WhenCMV-fl was compared with the TSTA system, the FL activity by using theTSTA system was observed to be 2- to 3-fold lower than that driven bythe CMV promoter. A comparison of fl expression by using both theone-step and the TSTA systems reveals a ˜50-fold gain (P<0.01) with theTSTA system when compared with the one-step system (FIG. 2A). Todetermine whether the TSTA system-mediated amplification is restrictedto prostate cells alone, we studied the levels of fl expression in twonon-prostate cell lines, C6 and HeLa. Firefly luciferase reporter geneexpression after androgen administration is minimal in both the celllines tested, indicating the tissue-specific nature of the PSE promoter(FIG. 2B). The results indicate a strong stimulatory effect ontranscription exerted by the GAL4-VP16 fusion protein while maintainingtissue specificity.

TSTA System Mediates Prostate-Specific Amplification of HSV1-sr39tkExpression in LNCaP Cells, Demonstrates Cell-Type Specificity, andExpression Increases with Androgen Dose

To test the HSV1-sr39tk reporter system, we used the T5 reporterconstruct with PG for transient transfections in LNCaP cells. Theresults with HSV1-sr39tk were similar to that observed by using fl.After androgen treatment, HSV1-sr39tk expression mediated by the TSTAsystem is significantly greater (˜24-fold) (P<0.01) when compared withthe expression in cells transfected with the reporter template only (T5)(FIG. 3A). Further, the TSTA system-mediated amplification ofHSV1-sr39tk expression levels are ˜12-fold greater than those driven byPT alone (one-step) (FIG. 3A) in the presence of androgen. Although thegain in amplification for HSV1-sr39tk is lower than fl, it isnevertheless highly significant (P<0.01). To determine whether the TSTAsystem-mediated reporter gene amplification is cell-type specific, westudied the levels of HSV1-sr39tk expression in two non-prostate celllines, C6 and HeLa. HSV1-sr39tk expression level after androgenadministration is minimal in both the cell lines tested, demonstratingthat the TSTA system-mediated activation from the PSE promoter is highlycell-type specific (FIG. 3B). We further evaluated the effects ofandrogen response in LNCaP cells by using HSV1-sr39tk. In the absence ofandrogen, HSV1-sr39tk expression levels are similar to the valuesobtained by using the reporter template alone (minimal). Aconcentration-dependent increase in HSV1-sr39TK activity is observedwith increasing androgen concentration, indicating that androgentreatment greatly enhances the TSTA system-mediated activation from thePSE promoter (FIG. 3C).

TSTA System Mediates Prostate-Specific Amplification of Fl Expression InVivo

To further test the utility of the TSTA system in vivo, we injectedtransiently transfected LNCaP cells (transfected with PG and L5plasmids, L5 plasmid alone, and PL plasmid) i.p. in female nude mice.Optical CCD imaging of all mice 15 min after injection of transfectedLNCaP cells reveals basal levels of fl expression (RLU/min<100). Themice were scanned again 24 h and 48 h after the implantation of androgenpellets. The results of the 24-h imaging were similar to those observedat 48 h. Forty-eight hours after pellet implantation, the control mice(L5 plasmid alone) again show minimal levels of fl expression(RLU/min<100) (FIG. 4A). In the mice representing the TSTA system, flexpression is observed to be much higher (RLU/min ˜500) when comparedwith the control and the one-step system (RLU/min<100) (FIG. 4B). Micerepresenting the TSTA system that did not receive androgen pelletsdisplayed basal levels of fl expression at 48 h (FIG. 4A). This isindicative of the specificity of androgen in activating the PSE promoterin vivo. The results were found to be reproducible between experimentalgroups. The induction of transcriptional activation upon androgenadministration across five mice in each of two groups is illustrated inFIG. 5. The TSTA system-mediated amplification in fl expression shows a˜5-fold gain when compared with the one-step system (P<0.05). Theseresults demonstrate the ability to image in vivo the expression of fldriven by a weak, tissue-specific promoter in a mammalian system byusing the TSTA approach. The TSTA system mediates tissue-specificexpression of fl in a mammalian system.

Discussion

We have previously demonstrated that adenoviral-mediated HSV1-tkexpression can be non-invasively imaged by using acycloguanosine analogsand microPET (1, 14). We have been evaluating methods to non-invasivelyimage reporter gene expression driven by weak promoters such as the PSEpromoter. Several of the known weak promoters also demonstrate tissuespecificity (33). In the present study, we describe a strategy toovercome the weak transcriptional activity of the PSE promoter for usein tissue-specific imaging applications. We used a two-steptranscriptional amplification (activation) approach where, in thepresence of androgen, a relatively weak PSE promoter activates aGAL4-VP16 fusion protein, which, in turn, drives reporter geneexpression under the control of GAL4 response elements in a minimalpromoter.

In the present study, we observed the TSTA system-mediated amplificationof fl expression to be 50-fold higher than the one-step system in LNCaPcells in the presence of androgen. We have also demonstrated the TSTAsystem to be highly tissue-specific. Similar results were obtained whenwe changed the reporter gene from fl to the HSV1-sr39tk, although thefold gain in HSV1-sr39tk expression levels was lower than f. Thedifferences in degree of amplification between fl and HSV1-sr39tk may beattributed to different levels of mRNA amplification and differences inthe two enzymes to act on their corresponding substrates. The TSTAsystem, however, does lead to a statistically significant increase inamplification for both the fl and HSV1-sr39tk in a tissue-specificmanner.

The TSTA system-mediated activation of the PSE promoter depends highlyon the levels of androgen as evidenced by the dose-dependent increase inHSV1-sr39tk expression. The system seems to function in a continuousmanner (as opposed to a binary on/off fashion). There also does not seemto be a threshold effect. As more androgen becomes available, levels ofGAL4-VP16 fusion protein increase, thereby increasing the ability tobind to GAL4 binding sites on the reporter template, resulting ingreater levels of fl and therefore greater imaging signal. Finally,although we did not see full saturation, higher levels of GAL4-VP16 havepreviously been reported to inhibit transcription (referred to assquelching) (30, 34, 35).

We further tested the utility of the GAL4-VP16 induction system in vivoto non-invasively image tissue-specific amplification of reporter geneexpression. To validate this system in vivo, several issues neededconsideration. These included the development of (i) LNCaP andnon-prostate cell lines stably expressing both the effector and reporterconstructs and (ii) construction of adenoviral vectors containing thetwo components of the TSTA system. Both of these approaches requireconsiderable time before they can be tested in vivo. To expedite theprocess of in vivo evaluation, we injected transiently transfected LNCaPcells (using fl as the reporter gene in the absence of androgen) infemale nude mice. The mice were imaged by using a sensitive cooled CCDcamera before and after implantation of testosterone pellets. All micedisplayed basal levels of fl expression in the absence of androgen.Twenty-four to 48 h after androgen administration, the mice representingthe TSTA system showed a significantly greater level of fl expressionwhen compared with the control and one-step mice. We observed similarhigh levels of induction across several mice with a ˜5-fold gain for theTSTA system over the one-step system. In fact, in the absence of theTSTA system, the fl expression was close to background, and there-forecells could not be imaged. These initial results are aimed atnon-invasively imaging reporter gene expression in a mammalian system byaugmenting the transcriptional activity of a weak promoter by using theTSTA system. The level of TSTA amplification in vivo depends on thepharmacokinetics of androgen availability to cells, and it is possiblethat the in vivo signal can potentially be higher than observed based onthe imaging time after androgen induction. Future studies will need toaddress the exact correlation between levels of androgen in blood andthe levels of induction in vivo.

Fang et al. (36) have examined the functionality of the GAL4-VP16transactivator to evaluate phosphoglycerate kinase (PGK) promoteractivities in vivo by using adenoviral vectors. Nettelbeck et al. (33)have used recombinant transcriptional activation to establish a positivefeedback loop initiated by transcriptional activation from a vonWillebrand factor promoter. The GAL4-VP16 fusion protein was used toachieve target gene amplification from a prostate-specific antigenpromoter (37). Both of these approaches were targeted at increasing theexpression of transgenes for use in cancer gene therapy protocols.Further, the GAL4-VP16 responsive TSTA system has been recently used inconjunction with zebrafish tissue-specific promoters with greenfluorescent protein reporters (35). In this study, the GAL4-VP16 wasinjected transiently into fish embryos that developed with minimaltoxicity, permitting imaging of specific tissue in adult fish.

It is important that any reporter gene-imaging approach notsignificantly perturb the cells/animal models being studied. There ispotential for the GAL4-VP16 system to be toxic to cells (35, 38). Inzebrafish, low levels of injected GAL4-VP16 were apparently notdeleterious to development, whereas higher levels were (35). However, inthe current study, only transiently transfected cells were used, sotoxicity is not practical to detect. Future studies with stable celllines, gene therapy vectors, and transgenic animal models will requirestrict characterization of potential toxicities of the TSTA approach. Itmay be the case that levels of GAL4-VP16 will need to be modulated tostrike a balance between amplification and any potential toxicity.

The approaches validated in the current study should lead to bettervectors for imaging gene therapy, study of tumor growth and regressionfollowing pharmacological intervention, as well as development oftransgenic models with enhanced reporter gene expression. Applicationsand extensions of the TSTA approach include: (i) imaging PET reportergenes (e.g., HSV1-sr39tk) and other in vivo reporter genes; (ii)enhancing reporter gene expression by modifying the regulatorycomponents of the PSE promoter and building a single vector thatincorporates both components of the TSTA system; (iii) replacement ofthe PSE promoter with other weak promoters, thereby enabling one totarget site-specific genes in vivo; (iv) the study of multipleendogenous genes by driving expression of GAL-4-A from one promoter andB-VP-16 from a separate promoter where genes A and B can be chosen sothat their respective proteins interact (this procedure should allow theexpression of a reporter gene if and only if both promoters areactivated); (v) imaging of protein-protein interactions in vivo usinginducible two-hybrid mammalian expression systems; and (vi)amplification of both therapeutic and reporter genes by modifying thereporter template.

EXAMPLE 2

The following provides descriptions of molecular engineering of atwo-step transcription amplification (TSTA) system for transgenedelivery in prostate cancer.

The concept of tissue specific gene therapy and imaging has beenhampered by lack of specificity, borderline efficacy and inadequatedelivery methods. Improvements in all three areas must occur totranslate the promise of gene therapy into meaningful clinicalapplications [39]. In prostate cancer, the most common strategy tocontrol specificity of transgene expression is to employ the regulatoryregion of a prostate gene product such as the prostate specific antigen(PSA) (reviewed in [40]). In order to achieve optimal therapeuticefficacy, one goal of the field has been to achieve prostate specificpromoter activities similar in magnitude to those of ubiquitously activeviral enhancers such as Simian Virus 40 (SV40) and Cytomegalovirus (CMV)[41]. A logical and systematic approach to improve the PSA regulatoryregions for combating prostate cancer involves several steps. Novelpromoter constructs are engineered and their activity in cell-basedtransfection studies is evaluated. The most promising constructs arecloned into recombinant adenoviral vectors or other efficient in vivogene delivery vehicles) and tested in pre-clinical models [42, 43].

Our labs have recently initiated such studies for prostate cancer. Weinitially reported a strategy to augment the specificity and activity ofthe PSA enhancer by exploiting the synergistic nature of androgenreceptor (AR) action. The key regulatory elements of the PSA enhancerinclude a proximal promoter (−541 to +12) comprising two binding sitesfor the androgen receptor (AREI and II) and a distal enhancer, whichcontains a 390-bp androgen-responsive core region [44, 45]. The coreregion contains a cluster of closely spaced androgen response elements(AREs) and sites for other transcription factors [46, 44, 47, 45, 48,49]. The enhancer is active in both androgen-dependant (AD) and androgenindependent (AI) cancer cells [49]. AR binds cooperatively to theenhancer and mediates synergistic transcription [48]. Other factorswithin and outside of the enhancer contribute to prostate specificity[50-52, 49]. We found that molecular engineering of the PSA enhancer byduplication of the core or fusion to multiple AREs generated 20-foldhigher activities than the parental constructs yet retained androgeninducibility and tissue specificity [53]]. Furthermore, we showed thatthe enhanced activity, inducibility and specificity of the chimericconstructs were maintained in an adenoviral vector expressing fireflyluciferase (FL) [53].

The two-step transcriptional activation system (TSTA) is anotherstrategy for augmenting PSA transgene expression [54]. In the TSTAsystem, a potent transcriptional activator, which is driven by acell-specific promoter, acts on a second expression plasmid, whichencodes the reporter/therapeutic protein. This two-step approach resultsin cell-specific amplification of expression. The activator is oftenGAL4-VP16, a fusion protein comprising the DNA binding domain from theyeast transcription activator GAL4 and the activation domain from theHerpes Simplex Virus 1 activator VP16. GAL4-VP16 assumes a uniquespecificity and potency that does not naturally exist in the mammaliancells [55, 56]. The cell specific TSTA approach is based on the original“enhancer trap” methodology employed in Drosophila to studydevelopmental regulation of gene expression [57, 58].

In one application of the TSTA approach to prostate cancer, Segawa andcolleagues attached the 5.3-kb intact PSA regulatory region to GAL4-VP16and demonstrated androgen-dependant activation of a FL reporter genebearing upstream GAL4 binding\sites and ablation of cancer cells using aTSTA-driven toxic gene [59]. Our groups employed a similar TSTAexpression approach but coupled it to imaging of prostate cancer inliving mice. Prostate cancer cells injected into live mice werevisualized using a cooled Charge Coupled Device (CCD) optical imagingcamera by using a streamlined 2.4-kb PSA enhancer-promoter attached to aFL reporter, [60]. In both cases, the TSTA system maintained androgenresponsiveness, yet substantially amplified the signal versus thereporters driven directly by the PSA regulatory region.

In the current study, we sought to optimize the TSTA system formolecular imaging with eventual applications to therapy. Noninvasivemolecular imaging with reporter genes can be used for imaging tumors andmetastases, to monitor the therapeutic efficacy of drugs, celltrafficking, gene delivery and expression, as well as the study ofvarious transgenic models [61]. Our objective was to augment thepromoter activity and generate a titratable system. Augmented activitywill be required to transition the FL-based CCD approach to clinicallyrelevant methodologies such as positron emission tomography (PET).Titratability of expression, on the other hand, is an importantsafeguard in gene therapy. Often, the untoward side effects ofintroducing and expressing exogenous genes in animals and patients cannot be fully predicted by in vitro or tissue culture experimentation. Inaddition, titratability is necessary because tumors and cell linesdisplay varying AR responses [62, 63]. For example, biphasic effects ofandrogen on AR activity have been observed and excess levels of androgeninhibit PSA production and cell growth [64]. A study on the LNCaPsubline also suggests that chronic androgen treatment induces reversiblecell adaptation to the stimulant [65]. An additional complexity is thatexcess GAL4-VP16 exhibits a transcriptional inhibitory effect calledsquelching [66], which can generate cell toxicity and might confoundTSTA under certain conditions (see, for example, [67]).

The concept of adjusting transgene expression by controlling theactivator and promoter potency is detailed in older studies by one of uson GAL4-derived activators [56]. In a subsequent study, a wide range ofactivities was generated by exploiting the synergistic behavior observedby varying the number of activation domains on the GAL4-derivedactivator and GAL4 binding sites on the reporter plasmid [68]. Here wecombine the modified GAL4 system with natural and chimeric PSA enhancersto create a highly active, robust expression system for prostate cancergene imaging and therapy. We demonstrate the cell specificity, efficacyand utility of this system by imaging living mice using cooled CCDoptical technology.

Results

The Chimeric TSTA System

We outline the TSTA rationale in FIG. 6A. There are four primaryvariables in our system: i. The potency of the prostate specificpromoter driving GAL4-VP16 (the effector) (FIG. 6B); ii. the number ofGAL4 binding sites proximal to the FL reporter gene (FIG. 6C); iii. thepotency of the GAL4-VP16 derivative (FIG. 6B); and iv. The presence ofthe effector and reporter genes on the same plasmid (FIG. 6D). Eachvariable offers a unique opportunity to modulate gene expression.

We employed two variants of the tissue specific PSA promoter (FIG. 6B).The first is PSE, which contains a 2.4 kb fragment (−5824 to −2855),encompassing the 390 bp core PSA enhancer core region (−4326 to −3935),linked to the proximal PSA promoter (−541 to +12). The second, PSE-BC([53], abbreviated as PBC here), contains the PSA enhancer with aduplicated 390-bp core but a deletion of an 890 bp intervening sequencebetween the enhancer and promoter (−3743 to −2855). These modificationsaugmented androgen-responsive expression 20-fold in cell culture [53].

The reporter templates contain the FL gene under the control of 1, 2 or5 copies of the 17 bp GAL4 binding sites positioned 23 bp upstream of aminimal promoter containing the adenovirus E4 gene TATA box (FIG. 6C)[68]. The resulting plasmids are termed G1-L, G2-L and G5-L. We used PSEand PBC to express recombinant GAL4-VP16 variants to generate a seriesof effector plasmids that display a gradient of activities (FIG. 6B).PSE expresses the 147 amino acid GAL4 DNA binding domain (DBD) bearing asingle copy of the 42 amino acid VP16 activation subdomain (amino acids413-454), PSE-VP1 [68]. We engineered PBC to express GAL4 DBD fusionproteins containing 1, 2 or 4 copies of the VP16 subdomain. Weabbreviated the resultant plasmids as PSE-VP1, PBC-VP1, PBC-VP2 andPBC-VP4 and cloned the optimal combination of PBC-VP2 and G5-L into asingle plasmid termed PBCVP2G5-L.

We expressed the parental plasmids GAL4-VP1, -VP2 and -VP4 from the SV40enhancer as positive controls to provide a benchmark for comparison. Asan additional benchmark, we used a FL construct driven by the CMVenhancer termed CMV-L. We systematically evaluated the TSTA constructsby co-transfection assays into the androgen-responsive prostate cancercell line, LNCaP, in the presence of 10 nM R1881. We normalized eachexperiment using either CMV-L or the SV40 constructs and graphedrepresentative individual experiments. Subsequently, we tested severalcell lines to evaluate cell specificity. For ease of comparison with theTSTA system, we will refer to PSE or PBC directly driving FL expressionas the one-step system.

Evaluation of the Variables

We evaluated the variables to determine i. the relative efficacies ofthe one-versus the two-step systems; ii. the use of PSE versus PBC todrive GAL4-VP16 expression; iii. the effect of varying the number ofGAL4 binding sites; and iv. the effect of varying the number of VP16activation domains.

The TSTA system displayed enhanced activity and androgen inducibilityversus the one-step system in transfection assays. Co-transfection ofPSE-VP1 and G5-L resulted in an activated level in cell culture that was250-fold greater than the PSE-L one-step construct (FIG. 7). Further,the TSTA system retained strong androgen-responsiveness (compare + and−R1881). However, the PBC-VP1/G5-L combination exhibited an inducedactivity only 15-fold better than the PBC-L. Comparison of PSE-VP1 andPBC-VP1 on G5-L revealed only a 1.5-fold difference, which is notconsidered significant (P=0.1). This value was much less than the20-fold difference observed between PSE-L and PBC-L [15] suggesting thatdespite the augmented potency of PBC, the FL levels in LNCaP cells beginto saturate with G5-L as a reporter.

Variation of the number of GAL4 binding sites contributed to thetitratability (FIG. 8). The activity with PSE-VP1 increased nearly 8fold after increasing the number of sites from one (G1-L) to two (G2-L).However, raising the number of sites from two (G2-L) to five (G5-L)increased the activity an additional 60-fold. The pattern changed whenexamining the combination of a stronger promoter, PBC, and a more potentactivator, GAL4-VP4. In this example, the largest increase in activity,30-fold was observed from G1-L to G2-L. From G2-L to G5-L there was onlya 15-fold increase. The activity appears to saturate at five GAL4 sitesbecause G9-L exhibited the same activity as G5-L with PBC-VP2 as aneffector. In summary, PSE-VP1 and PBC-VP4 both increase transcriptionactivation synergistically as the number of activator binding sitesincreases. The magnitude of the synergy and the absolute level ofactivation, however, are related to the potency of the effector plasmid,providing a strategy to further adjust the expression levels in thissystem.

The final parameter we measured was the effect of multimerizing the VP16activation domain (FIG. 9). With G5-L as reporter, PBC-VP2 displayed a3-fold greater activity than PBC-VP1 (P=0.0007). Surprisingly, PBC-VP2is modestly better than PBC-VP4 (P=0.02), possibly because PBC-VP4 iscausing an inhibitory phenomenon called squelching [66]). Nevertheless,the hallmark was the combination of PBC-VP2 and G5-L, where the level ofactivated expression reproducibly exceeded the activity of bothbenchmarks (CMV-L and the combination of SV40-VP4 with G5-L). FIG. 10illustrates the full spectrum of expression levels observed in oursystem, with an 800-fold variation from the weakest one-step to thestrongest two-step system.

Tissue/cell Specificity of the TSTA System

We chose the TSTA combination of PBC-VP2 and G5-L for analysis of tissuespecificity (FIG. 11). We compared prostate and non-prostate lines, andcell lines expressing and lacking AR to assess authentic prostatespecificity and distinguish it from simple androgen responsiveness. TheLAPC4 cell line is an advanced prostate cancer cell (tumor cells frompatient with refractory/metastatic prostate cancer) line that expressesAR and displays modest androgen-responsiveness as measured by PSAinduction [69]. The mammary carcinoma cell line MCF-7 also expresses ARbut not PSA. The human cervical cancer cell line HeLa cell is negativefor AR and PSA. fAR-HeLa expresses flag-tagged human AR but not PSA[48]. Finally, we included the human hepatic cell line HepG to test forpotential activity in liver.

During our initial tests we discovered that CMV-L responds to androgenstimulation (FIG. 9). We employed SV40-driven GAL4-VP2 and G5-L as abenchmark to properly normalize experiments in different cell types. TheSV40-VP2/G5-L displayed a similar activity as CMV-L in LNCaP and was notsignificantly affected by androgen in any cell lines tested. In LNCaPcells, we consistently observed that, in the presence of androgen, thecombination of PBC-VP2 and G5-L had an activity that was similar to thatof SV40-VP2 and G5-L. This observation was consistent withimmunoblotting results (FIG. 11, panel B) where PBC and SV40 expressedGAL4-VP2 at similar levels. However, PBC expression wasandrogen-dependent whereas SV40 expression was not influenced byandrogen.

As shown in FIG. 11 we found that the TSTA system displayed activitiescomparable to the SV40 benchmark only in the prostate cancer cell lines,LNCaP and LAPC4 (>90% and 75%, respectively). In contrast, we found thatthe activities of the TSTA system relative to the benchmark were 0.2% inHepG cells, 0.7% in HeLa cells and 1.5% in MCF-7 cells. In a systemwhere AR was overexpressed, fAR-HeLa cells, the TSTA system elicited 2%the activity of our benchmark. The difference between HeLa and fAR HeLawas significant (P=0.4).

The Effector and Reporter on the Same Plasmid

A single plasmid bearing both the effector and reporter greatlyincreased activity but maintained cell selectivity (FIG. 12). Theconstruct, referred to as PBCVP2G5-L, contains a 3.5-kb PBC-VP2 NotIfragment inserted into the G5-L (FIG. 6D) in a “head to head”orientation, with 170-bp between G5 and the 5′ end of the distal copy ofthe PSA core enhancer. Transfection of the single construct PBCVP2G5-Linto LNCaP cells yielded an activated FL activity 10-fold higher thanthat seen with the same molar amount of the two-construct PBC-VP2/G5-LTSTA system. The single construct did not display as much activityrelative to the benchmark in LAPC4 cells, possibly due to the reducedandrogen dependency of LAPC4. Nevertheless, PBCVP2G5-L maintained tissueselectivity as illustrated by its low activity in HeLa, fAR-HeLa, HepG2,and MCF7 cells. When we cloned the PBC-VP2 NotI fragment in the“head-to-tail” orientation with G5-L to generate a single construct, wediscovered that PBCVP2G5-L(R) displayed greatly reduced activity versusthe head-to head orientation. We do not understand the cause of thiseffect.

Application of the Chimeric-TSTA System to Imaging FL Reporter GeneExpression in Living Mice

We show that the chimeric-TSTA system displays robust expression inimaging studies in live mice. To validate the potential of the modifiedTSTA for imaging we determined whether the differences in activity couldbe reproduced qualitatively in an animal imaging system. A cooled chargecoupled device (CCD) camera converts bioluminescent photons toquantifiable electronic signals. The luminescence is recorded andgraphically displayed by superimposing a colored topographic pseudoimageon a photograph of the animal. Previous studies had established thatwithin the proper time frame, the RLU signals acquired by the camera arelinear to exposure time and amount of FL activity as assayed byluminometry [70]. We transfected LNCaP cells or HeLa cells with ouroptimal TSTA constructs, treated with R1881, and implanted the cellssubcutaneously onto the dorsal surface of the mice. We separated themice into 4 groups. Within each group we injected three types oftransfected cells. To eliminate positional artifacts, we performed theexperiments in triplicate; we rotated each group of cells through allthree implantation sites. FIG. 13 shows pictures of representative micefrom each group. In group 1 (FIG. 13A), the G5-L negative control showedno detectable signal and the signal generated by PSE-L was barely abovethe background. In contrast the signal for the one-step vector PBC-L wasevident and equal to 800 RLU/min at the maximum intensity. When weanalyzed the two-step system (FIG. 13B), signals of over 6500 RLU/minwere obtained with the optimal two-construct TSTA system.

The signals appeared to be both ligand- and cell-specific. HeLa cellstransfected with PBC-VP2/G5-luc (FIG. 13C), or LNCaP cells grown withoutR1881 did not display a signal above background. Remarkably, weestimated the maximum signal from PBCVP2G5-L, the single construct, tobe 55,000 RLU/min, which is nearly 10-fold greater than the signalgenerated by the two-construct TSTA system (FIG. 13C). CMV-luc exhibiteda similar signal in both LNCaP and HeLa cells. In summary, we havepresented here our initial effort to apply the augmented TSTA system tomolecular imaging and we discuss the potential applications of thissystem below.

Discussion

Use of the Chimeric TSTA System to Modulate Expression

We have modulated the activity of the TSTA system by introducing potentPSA enhancers and potent derivatives of GAL4-VP16. Remarkably, thelargest increases in activity came not from increasing the potency ofthe PSA promoter but from either increasing the potency of the activatoror by placing the TSTA components on a single plasmid. As shown in FIG.8, increasing the number of activator binding sites from G1 to G5greatly amplifies the activity by 240 to 450 fold, depending on theactivator expressed (PSE-VP1 or PBC-VP4). Moreover, duplication of theactivation domain from PBC-VP1 to PBC-VP2 resulted in a 3-foldenhancement of activity. These results reinforce the concept ofsynergistic activation of transcription and its use in varying theactivity of an expression system.

We observed the single most dramatic increase in activity by placing theTSTA system on a single plasmid. Comparison in LNCaP cells of the singleconstruct, PBCVP2G5-L, with the combination, PBC-VP2 and G5-L, revealeda striking 10-fold difference. The positioning of the two components onthe same DNA molecule may generate a feed forward loop, where GAL4-VP16drives G5-L and raises its own expression level by binding upstream ofPBC. Similar loops have been described [71]

There are numerous combinations that could still be tested. The effectsof the PSE-VP2 or -VP4 series have not been compared with PBC-VP2 and-VP4. Additionally, certain conditions, such as the R1881 concentration,have not been adjusted; this was performed in an earlier study [60].However, the values will likely fall within the ranges we observed. Thestudy has indicated that we have reached, or are close to, the limit ofamplification using the present TSTA components.

Others have explored the concept of TSTA in imaging and gene therapy.The Fraser group used a TSTA system, which employed a GFP effector, andachieved enhanced, tissue specific bioluminescent signals to study Zebrafish development [67]. Segawa and colleagues combined the PSA wild typeenhancer-promoter with GAL4-VP16 for gene therapy in prostate cancer[59] and our groups recently employed an early version of TSTA inimaging.

Remarkable progress has been reported by others, who have explored theoptimization of prostate specific promoters. For example, the prostatespecific PSA and human Kallikrein 2 (hK2) promoter/enhancers have beenmodified to generate potent androgen-responsive expression systemsanalogous to our chimeric enhancers [72, 73]. A direct comparison of thestudies is difficult because of differences in enhancers, reporters (GFPvs. FL), minimal promoters, positive benchmarks (different CMVconstructs) or methods of DNA transduction. We emphasize that our studywas geared towards the same endpoint as the others but we focused onincreasing potency and signal flexibility for use in imaging andtherapy. By employing transient transfection assays we demonstrated theamplitude, titratability and specificity of our system over an 800-foldrange. We tested certain points within this range and they maintainedthe same approximate activities by a cooled CCD imaging approach. Thestrongest constructs repeatedly exceeded the activities of multiplebenchmarks.

Activity, Inducibility and Specificity

The current methodology prohibits us from drawing conclusions about theabsolute degree of androgen inducibility. Basal expression increased asthe activity of the system increased from the one-step to the mostpotent two-step constructs. We believe that much of the increase inbasal expression is a consequence of incomplete depletion of steroidsfrom the cultured cells. The argument is based on two observations: i.The basal activity of the PSA enhancer constructs in charcoal-strippedserum can be further reduced by addition of the anti-androgen casodex.ii. The small amount of residual androgen in charcoal-stripped mediumwill likely result in the same amount of activator being synthesizedwhen comparing different GAL4-VP16 derivatives expressed from a similarpromoter such as PBC. This point is supported by our result showing thatthe 3-fold increase in potency of activators, i.e., PBC-VP1 vs. PBC-VP2,is observed both in the presence and absence of R1881. Nevertheless, thesystem still displayed cell type specificity. Ultimately, the mostrigorous test of true androgen responsiveness and tissue specificexpression will be to perform transgenic animal studies or to employviruses stably expressing the TSTA system to infect xenografts or otheranimal models; this is the current focus of our research.

Applications to In Vivo Imaging

The cooled CCD optical imaging belongs to a new generation of in vivoimaging technologies that use fluorescent or bioluminescent reportergenes to produce a signal from within a living animal. The CCD approachdetects low levels of luminescence consistently and reproducibly fromfur-covered animals without the need for an external light source. [74,70].

Luciferase monitoring of transgene expression is still in its infancyand is limited to small animals due to significant absorption andscatter of visible light within the animal [70]. Positron emissiontomography (PET) is a technology currently employed in clinical settings[75-77]. PET utilizes molecular probes labeled with positron emittingisotopes and produces dynamic signals measurable by a circular array ofdetectors. PET generates tomographic images reflecting the concentrationand location(s) of probes in a living subject of any size. SuccessfulPET imaging has been performed with the Herpes Simplex-Virus-1 (HSV-1)Thymidine Kinase (TK) reporter gene expressed from a CMV enhancer [75,77]. TK utilizes ₁₈ F-labeled ganciclovir/penciclovir to generate asignal primarily in cells expressing the reporter gene. However, forsome applications PET may have a lower sensitivity than bioluminescenceimaging [70]. A significant improvement in sensitivity, in order toimage the least number of cells, is necessary to make reportergene-based PET methods viable for imaging prostate cancer progression inpre-clinical and clinical models. The modifications reported in ourcurrent system are aimed at improving a prostate-specific PET-basedreporter gene methodology.

Extensions of the TSTA System

The TSTA system can be manipulated for use in targeting cancer indifferent ways. New cell and promoter specific regulatory elements canbe introduced to further up- or down-regulate expression [54].Replacement of the VP16 activation domain with domains responsive tounique signals can be employed to identify novel cellular signalingpathways. Furthermore, other prostate cancer promoters can be utilizedand novel reporter or therapeutic genes can be added. These additionswill increase the utility and regulatability of TSTA. Although androgenand AR are central to early prostate cancer progression, later stages ofproliferation gradually utilize cross-talk among AR and signalingcascades including MAPK, PI3K, PKA, EGFR, IGFR and TGF-β [78, 79, 69,80, 81]. The concept of TSTA can be readily applied to understanding theramifications and activity of these cellular pathways during cancerprogression.

In conclusion the major advances of TSTA are its cell selectivity,activity and robustness relative to cell specific promoters. Itsflexibility will permit widespread utility in cancer research.

Materials and Methods

Cell Culture

We grew the human prostate cancer cell line LNCaP in RPMI 1640supplemented with 10% Fetal Bovine Serum and 1% Penicillin/Streptomycinsolution. We grew HeLa, fAR-HeLa [48], and the human hepatic line HepGcells (ATCC) in DMEM with 10% Fetal Bovine Serum and 1%Penicillin/Streptomycin. We grew the human prostate cancer line LAPC4[69] in IMDM (GIBCO) with 10% Fetal Bovine Serum and 1%Penicillin/Streptomycin. Prior to transfection, we transferred the cellsfor 24 hours into media that contains 5% charcoal stripped serum.

Plasmids

The baseline PSA promoter construct termed PSE and the chimeric PSE-BC(abbreviated here as PBC) are as previously described [53]. To constructpPSE-VP1, the HindIII-XbaI fragment was excised from pSV40-VP1 andinserted downstream of the 2.4 Kb PSA promoter PSE. We generated theother effectors, PBC-VP1, PBC-VP2 and PBC-VP4 using similar strategies[53]. We constructed G1, G2 and G5-L as described [60]. We cloned theNotI Fragment bearing the PBC promoter and GAL4-VP2 gene into the NotIsite of G5-L generating a single vector termed PBCVP2G5-L that containedboth components of the TSTA system.

Cell Transfection

On day 1, we plated LNCaP cells in 6-well plates in RPMI 1640 containingcharcoal stripped FBS. We performed transient transfections 24 hourslater using Tfx-50 (Promega) with a lipid:DNA ratio of 4:1. Eachtransfection mixture contained 0.5 μg of the effector and reporterplasmids or reporter plasmid alone with pGL3B carrier DNA.Methylenetrienolone (R1881; NEN Life Science Products, Boston, Mass.)was added to the medium at a concentration of 10 nM/well one hourfollowing transfection, and the cells were incubated for 48 h. The cellswere harvested and lysed using the passive lysis buffer provided in theassay kit for measuring FL activities (Dual-Reporter Luciferase AssaySystem, Promega). FL activities of 5% of the cell lysates with 100 ml ofsubstrate D luciferin were measured using a luminometer (Lumat 9507,Berthod Germany) with an integration time of 10 sec.

Immunoblot Analysis of GAL4-VP16 Expression

LNCaP cells were grown in 10-cm dishes and transfected with selectplasmids expressing the TSTA components. We harvested and lysed thetreated cells using RIPA lysis buffer (10 mM Tris-HCl, 150 mM NaCl, 0.1%SDS, 1% DOC, 1 mM EDTA and 1% NP40). We normalized extracts by proteinconcentration (Bio-Rad Dc protein assay Kit), the samples werefractionated on 4-15% gradient acrylamide gels (Bio-Rad) and subjectedto immunoblot analysis with rabbit polyclonal antibodies generatedagainst intact GAL4-VP16 [82].

In Vivo Studies

We treated transiently transfected LNCaP and HeLa cells with 10 mMR1881, harvested 40 hours post-transfection and resuspended in phosphatebuffered saline (PBS). We anesthetized female nu/nu mice with 40 μl ofketamine-xylazine (4:1) solution. To allow time for tissue distribution,a solution of D-luciferin (Xenogen, CA) in PBS (200 ml, 15 mg/ml) wasinjected into the peritoneal cavity prior to implanting cells. After 5min, 1×10₆ cells were suspended in 50 μl PBS, combined with 50 μl ofMatrigel (BD Biosciences, Bedford Mass.) and injected subcutaneouslyonto the dorsal side of the mice. Each mouse bore injections at 3 sites.Twenty minutes after intra-peritoneal injection of D-luciferin, weimaged the mice using a cooled CCD camera (Xenogen IVIS, Xenogen Corp.,Alameda, Calif.). At the time of injection into animals, an aliquot ofthe cells was also analyzed for FL activity using a luminometer asdescribed above. All studies were performed with full approval from theUCLA Animal Research Committee (ARC).

CCD Imaging and Quantitation

We placed the mice prone in a light-tight chamber and a gray scalereference photograph was obtained under low-level illumination. Wecollected photons emitted from within the mouse and transmitted throughtissue and integrated them for an acquisition time of 1 to 5 min. Weobtained images and analyzed them using Living Image Software v4.02 A(Xenogen Corporation, Alameda, Calif.) v2.11 and Igor Image AnalysisSoftware Wavemetrics, Seattle, Wash.). We drew regions of interest (ROI)over the visible light signal to quantitate the light. We normalized themaximum relative light unit (RLU) signals measured to acquisition timeto obtain maximum (RLU/min). We calibrated the system as describedearlier [70].

EXAMPLE 3

The following provides descriptions of visualization of advanced humanprostate cancer lesions in living mice by a targeted gene transfervector and optical imaging.

Continued improvements in screening, early detection, and treatment oflocalized disease have led to a steady decline in prostate cancermortality over the past ten years (83-85). Despite these advancements,prostate cancer continues to be the second highest cause of cancerdeaths in American men. Endocrine therapy, employing castration and/oranti-androgens, is the only effective treatment for advanced, metastaticdisease (86, 87). Even though these patients continue endocrine therapy,prostate cancers invariably relapse within a mean time of 18-36 months(88), after which they are considered androgen-independent (AI), and areunresponsive to existing treatments. Vector-based prostate delivery oftherapeutic transgenes represents a potential alternative or adjuvant toexisting therapies, such as chemo- or radiotherapy, for the treatment ofAI disease. As with other emerging therapies, vector-based approachesare challenged with achieving acceptable levels of efficacy and safety.Use of strong constitutive viral promoters, such as those ofcytomegalovirus (CMV) and Rous sarcoma virus (RSV), enables high levelsof therapeutic transgene expression, but could result in accompanyingdamage to healthy tissues (89). To improve the activity and specificityof prostate-targeted gene expression, we recently developed enhancedpromoters designed to augment prostate-specific transgene expression bymultimerizing key regulatory elements in the prostate-specific antigen(PSA) enhancer and promoter (90). One such promoter, PSE-BC, was 20-foldmore active than the native PSA enhancer/promoter in cell-based fireflyluciferase expression studies. Furthermore, when incorporated into anadenovirus vector (AdPSE-BC-luc), the promoter exhibited greatlyenhanced transcriptional activity in LNCaP prostate cancer cells andrestricted transgene expression in several non-prostate cell lines andmouse tissues (90).

To further advance the concept of prostate-targeted expression, wetested our approaches in several recently developed human prostatecancer (CaP) xenograft models, designated the Los Angeles ProstateCancer (LAPC) series, which was derived from clinical tissues obtainedfrom patients with advanced CaP and grafted into severe combined immunedeficient (SCID) mice. The models retain characteristics of clinicaldisease, including androgen receptor (AR) and PSA expression, therequirement for androgens, and metastatic potential (91, 92). One of themodels, LAPC-4, initially requires androgen for growth, but similar toclinical disease progression, tumors regress upon castration and an AItumor emerges (91). Demonstration of the utility of our approaches inclinically relevant models suggests that they may be applicable inclinical settings.

The application of a non-invasive imaging modality in a CaP-targetedgene therapy model may greatly aid in accurate assessment of in vivovector-mediated gene transduction and therapeutic efficacy. The recentlydeveloped cooled charged coupled device (CCD) camera for optical imagingis a sensitive approach for detecting bioluminescence emitted fromluciferin reacting with firefly luciferase in living animals (93-95).The advantages of a targeted gene transfer approach coupled withnon-invasive imaging include the ability to localize diseased tissue,and importantly, to accurately monitor the kinetics and levels oftransgene expression in diseased and healthy tissues. In this study, weemployed CCD imaging to achieve those ends in several mouse models ofhuman CaP.

Results

Discriminatory Expression Capability of a Prostate Specific GeneDelivery Vector

We first assessed the in vivo transcriptional targeting capability ofAdPSE-BC-luc in comparison to the strong constitutive AdCMV-luc (94).After systemic tail vein injection of either Ad, luciferase expressionwas monitored. The CCD signals are dependent on the administration ofD-luciferin substrate, as omission of luciferin results in weakbackground signals (FIG. 14A). The magnitudes of light signals (maximumrelative light units, RLU) are linearly proportional to the imageacquisition time (94). This important feature of cooled CCD cameraallows the signal intensity to be normalized to the acquisition time andbe reported as maximum RLU/min, represented by the color scale. Theacquisition times were reduced to offset strong signal intensities thatsaturate the CCD camera (e.g., liver signal in CMV cohorts; FIG. 14A).Despite comparable gene delivery to the liver in both groups (FIG. 14B),AdPSE-BC-luc-mediated expression in the liver was less than 10⁻⁵ that ofAdCMV-luc, as measured by CCD imaging and luminometry of tissue extractsfrom isolated livers (FIG. 14A and Table 1). These data confirmed thatAdPSE-BC-luc has high specificity for prostate tissue (90), as indicatedby its poor expression in liver, the major site of expression forAdCMV-luc. Furthermore, this non-invasive imaging modality correlateswell with current in vitro biochemical analysis of luciferase activity(94).

The prostate-specific transcriptional activity of AdPSE-BC-luc wasevaluated by direct injection of Ad into several human CaP tumorsimplanted into murine subcutaneous tissues in the lower back. Theresults from AD LAPC-4 models are shown in FIG. 15. The activity ofAdPSE-BC-luc in LAPC-9 tumors was similar to that observed in LAPC-4.Based on the activity seen 8 days post-injection, AdPSE-BC-luc displayed72-fold lower expression in AD LAPC-4 tumors than AdCMV-luc (FIG. 15 andTable 1). By comparing the ratio of activity in LAPC-4 tumors to that inlivers, AdPSE-BC-luc exhibited ˜1000 fold higher preferential expressionin CaP tumors than AdCMV-luc (FIGS. 14 and 15, and Table 1).

Kinetics of Transgene Expression in Living Mice Bearing Human ProstateTumors

One major advantage of the CCD non-invasive imaging system is theability for repetitive monitoring of Ad-based luciferase gene transferand expression in the same animal over time. Because the experiment wascarried out entirely in the same animal, and without any variation inthe genetic background or in gene delivery, smaller study cohorts couldbe used. Luciferase expression was evaluated in mice bearing AD LAPC-4tumors spanning a 3-week period after intra-tumoral injections ofAdCMV-luc (FIG. 15A) or AdPSE-BC-luc (FIG. 15B). The intra-tumoralsignals in the AdCMV-luc injected mouse (CMV1) displayed the highestactivity between 2 and 4 days post-injection, and diminished thereafter(FIG. 15A). Leakage of AdCMV-luc into the circulation afterintra-tumoral injections was observed in LAPC-4 tumors, as indicated bythe signals appearing in the liver (FIG. 15A). In fact, after 4 days,the liver signals exceeded those in the tumor, and gradually increasedover time (FIG. 15A and Table 1). The intra-tumoral signals continued todecrease to a level below the minimum scale of 1×10⁵ RLU/min at 15 dayspost-injection, whereas the liver signal remained robust at ˜1.5×10⁶RLU/min in this animal (CMV1).

By comparison with Ad-CMV-luc, the time course of intra-tumoralAdPSE-BC-luc expression was delayed (FIGS. 15A and 15B). The majority ofthe AdPSE-BC-luc injected tumors expressed negligible luciferase at 2days post-injection (3/5 mice). All 5 mice in the cohort start todisplay signals at 4 days, and peaked signals between 8 to 11 dayspost-injection (FIG. 15B and Table 1). The delayed course ofAd-PSE-BC-luc-mediated expression may be attributed to its lowertranscriptional activity relative to AdCMV-luc. In theAdPSE-BC-luc-injected cohort of 5 mice, only signals emitted from thetumors were detected at time points on or before 15 days post-injection(FIG. 15B). However, at 21 days post-injection, low-magnitudeextra-tumoral signals were visible (FIG. 15B) in 3 out of 5 animals.These low-magnitude signals (˜200 RLU/min; FIG. 15B) weredistinguishable from background luminescence because CCD imaging of thesame animal at 2 days post-injection, a time point just prior toinitiation of expression, revealed the background to be ≦70 RLU/min. Tolocalize the origin of light emission, we isolated organs from the BC4mouse and re-imaged at the time of sacrifice (FIG. 15C). The signals inthe upper chest and lower back emanated from the animal's lung andspine, respectively (FIGS. 15B and 15C).

Detection and Localization of Human Prostate Cancer Metastatic Lesions

We undertook detailed histological evaluation of the isolated organs todetermine whether metastatic lesions were in fact detected by ourmethodology. The elongated spinal signal was well-localized within thelength of the spinal column (FIG. 15C), so we processed this specimenfor further histological and immunohistochemical analysis. Highermagnifications revealed a large, elongated metastatic lesion embedded inspinal musculature, characterized by large pleomorphic nuclei and a highmitotic rate consistent with neoplasia (FIGS. 16A and 16B).Immunohistochemistry performed with an anti-human pan-cytokeratinantibody confirmed that the lesion was of human origin (FIGS. 16A and16B). The spinal lesion of another animal in the cohort (BC2) alsodemonstrated the same histological characteristics and a clearcorrespondence of CCD signal and lesion location at the caudal end ofthe spine (FIG. 16B). In another animal with lung signals (FIG. 16C), weprocessed the lung for immunofluorescent evaluation by confocalmicroscopy, using the human cytokeratin antibody. Evaluation of lungsections revealed specific cytoplasmic localization of the humanpan-cytokeratin antibody, which was identical to that seen in cells ofthe xenograft and metastatic spinal lesion (FIG. 16C). The staining wasundetectable in the lungs of non-tumor-bearing mice used as negativecontrols. Tumor cells in the lung were detected as micrometastaticnodules of 9 to 74 cells in several independent locations that occupied377 μm³ of the right lung and 46 μm³ of the left lung. The predominantlocalization of micrometastasis in the right lung of this animalcorresponded well with the CCD imaging result (FIG. 16C).

Elevated PSA-Based Expression in Advanced Androgen Independent Tumors

To determine whether AdPSE-BC-luc would be active in AI tumors grown inthe absence of testicular androgen, we first evaluated whether therewere obvious differences between either endogenous AR or PSA expressionin the AD and AI LAPC-4 xenografts. This result would offer insightsregarding the expression of the exogenously introduced luciferase gene.FIG. 17A shows that neither protein is down-regulated in AD and AItumors. In contrast, PSA protein expression appeared to increase in theAI subline. This up-regulated endogenous PSA expression paralleled theexogenously introduced luciferase expression mediated by AdPSE-BC-luc(FIG. 17B and Table 1). The CCD images of 3 animals from both AD and AILAPC-4 tumor-bearing cohorts, 11 days after intratumoral injection, areshown in FIG. 17B. At this time point, 9.8-fold higher activity wasobserved in AI as compared to AD tumors (FIG. 17B and Table 1). Unevenvector distribution and gene transfer are known to occur inintra-tumoral injections¹⁴. To rule out this limitation as a reason forhigher gene expression in AI versus AD tumors, we performed ex vivoinfection of single-cell suspensions derived from the AD and AIxenografts. Both CaP tumor cell types were easily infected by Ads, butthere was higher luciferase expression in AI than in AD LAPC-4 tumorcells (FIG. 17C).

Discussion

Hormone therapy for prostate cancer has changed little since itsintroduction 30 years ago, yet it continues to be the only effectivetreatment for advanced disease (97). As a result, gene-based therapeuticstrategies, covering a broad spectrum of approaches, have emerged aspromising alternatives or adjuvant to existing modalities, includingreplacement of defective tumor suppressors, cytotoxic enzyme-prodrugtherapy (suicide gene therapy), suppression of tumor-angiogenesis, andup-regulation of immune-mediated tumor surveillance (98). However, thedesign and application of vector-based cancer gene therapies mustaddress issues of both efficacy and safety.

Efficacy in vector-based gene therapy is dependent in part on theability of a vector to infect and transduce cells of the target tissue.Our results demonstrate that our prostate-specific Ad was capable oftransducing both the AD LAPC-4 and LAPC-9 prostate cancer xenografts.This study also showed that as LAPC-4 tumors progressed from AD to AI,the AI tumor cells continued to express AR, PSA, and the luciferasegene, despite the complete absence of testicular androgen (FIG. 17).Remarkably, not only was the PSA-based Ad transcriptionally active in AIprostate tumors, it displayed nearly 10-fold higher activity than in ADLAPC-4 tumors. As all of the models retain important features ofadvanced clinical disease (91, 92), these data support the possibilitythat a prostate-specific vector capable of transducing prostate cells inhuman patients can be developed to treat advanced disease.

One interesting observation that we noted in the AR immunohistochemicalanalysis was that positive AR staining appeared to be more diffuse andless nuclear localized in the AI than in AD tumor sections (FIG. 17A).This observation is consistent with our current understanding thatnuclear translocation of AR is mediated in part by androgen binding tothe receptor (99). When testicular androgen is depleted as in the LAPC-4AI model, AR nuclear translocation is impeded but not completelyinhibited (FIG. 17A). Both the mechanism of AR translocation and thefunctional role of nuclear localized AR in this setting may be key tounderstanding transcriptional regulation in AI prostate cancer cells.Due to the largely qualitative nature of immunohistochemical analysis weneed to fully characterize the AR localization and functional activityin the LAPC-4 model at the molecular level. However, many studiesprovide evidence that AR can function in AI disease. The evidenceincludes AR mutations that confer expanded ligand specificity (100), ARgene amplification and over-expression (101), cross-talk between othersignaling cascades and AR pathways (102, 103) and increased expressionof the nuclear receptor transcriptional co-activator, TIF2 (104). Infact, over-expression of Her-2/neu has been implicated in AI progressionof both the LAPC-4 model (103) and clinical cases (105). Regardless ofthe precise mechanism(s), a better understanding of the criticaltranscriptional regulatory pathways operative in advanced AI prostatecancer should enable the development of even more effective approachesfor targeting this disease in the future.

One prominent, serendipitous discovery that arose from our study wasdetection of metastatic lesions. Although the precise transductionmechanism of the metastatic lesions is unclear at this time, wepostulated that intra-tumoral injection of AdPSE-BC-luc leaked to thesystemic circulation, in a route similar to the case in AdCMV-luc, andinfected metastatic lesions. No discernable liver signal was detected inthe AdPSE-BC-luc cohorts, because the tissue specificity of the PSE-BCpromoter prevented expression in the liver. This hypothesis is supportedby our recent preliminary results showing that metastatic lesions can bedetected by systemic tail vein injection of AdPSE-BC-luc. Thealternative explanation for detecting metastasis could be thedissemination of a transduced cell(s) from the primary tumor. Thishypothesis is less likely, especially in the case of the large spinallesions (FIGS. 16A and 16B), because Ad-mediated expression is known tobe transient, and expansion from a single cell to the large lesion wouldlikely have resulted in loss of expression. However, lungmicrometastases could have originated from transduced cancer cells inthe xenografts.

Although CCD imaging can sensitively and specifically monitor luciferaseexpression in small animal models, it lacks the ability to providedetailed tomographic information (93-95). On the other hand, PositronEmission Tomography (PET) is a clinically utilized imaging modality thatcan provide quantitative, three-dimensional localization of imagingsignals. In fact, we have demonstrated that high-resolutionmicro-positron emission tomography (microPET) can track Ad mediatedherpes simplex virus thymidine kinase gene expression in the livers(106) and tumors (96) of living mice. Currently, studies to optimizeparameters of imaging of PET reporter transgene in human volunteers havebeen initiated (107). In preparation for the transition to clinicalapplications, it will be important to validate our vector-mediatedcancer targeting approaches utilizing microPET in animal models.Appropriate resolution of the issues discussed should continue toimprove the design, efficacy, and safety of prostate cancer gene-baseddiagnostic and therapeutic strategies.

Methods

Mice and LAPC Xenograft Propagation

SCID (scid/scid) mice were bred and maintained as previously described(91). Male mice of approximately 3 months of age were utilized in ourstudies. Mice were anesthetized as described (94) for all surgical orimaging procedures. LAPC-4 and LAPC-9 seed tumor cells from frozenstocks were generously provided by Dr. Charles Sawyers (91, 92).Xenografts were initiated by implanting ˜10⁶ viable cells subcutaneouslywith Matrigel (Collaborative Research, Bedford, Mass.), 50:50 by volumetotaling 100 μl. Once tumors were established, further expansion andpropagation of tumors were accomplished by implanting tumor fragments of1-2 mm³, and the AI sublines were grown and passaged several rounds insurgically castrated male mice (91). The viral injections were performedwhen the tumors reached a diameter of ˜5-7 mm, which required about 3-4weeks of tumor growth from the initial time of tumor passage. Singlecell suspension transient cultures of xenografts were generated asdescribed (92).

Adenoviral Vectors and Southern Hybridization

AdCMV-luc and AdPSE-BC-luc were generated as previously described (90,94). The Ads were titered by plaque assays on 293 monolayer cells (i.e.infectious units=plaque forming units). Total cellular DNA was extractedfrom the livers of two treatment cohorts at 4 days post Ad injection.The Southern blot conditions were as previously described (90). Theliver tissues were homogenized and lysed by proteinase K, and total DNAwas purified by phenol/chloroform extraction, followed by ethanolprecipitation. DNA was digested with Not I, subjected to agarose gelelectrophoresis. NotI restriction digestion liberated a 2.8 kb CMV-lucand a 4.6 kb PSE-BC-luc expression cassette. Non-radioactive digoxigenin(Roche Co. Germany) labeled luciferase DNA fragment was used as probe(90).

CCD Imaging to Detect In Vivo Luciferase Expression

The same total dose of 1.8×10⁹ infectious units were injected eithersystemically via tail vein or intra-tumorally. For intra-tumoralinjections, Ad were given in 6 doses of 10 μl each into 3 sites on 2consecutive days. The procedures for animal imaging studies wereperformed as described (94). At the specified days post-injection, theCCD images were acquired using the Xenogen In Vivo Imaging System(Xenogen IVIS™, Alameda Calif.), adapted with a cooled CCD camera. Imageanalysis was performed using IGOR software (Wavemetrics, Lake Oswego,Oreg.). The increase in RLU is linearly proportional to the increase inacquisition time (94). Therefore, we normalized the signal intensity tothe image acquisition time (RLU/min), such that an expanded range ofcomparative values beyond the saturation limit of 65,000 maximum RLUcould be achieved. The image analysis software can quantify signalintensities by integrating over a region of interest (ROI) representedby RLU/pixel/min. We found, however, that due to the great range ofsignal intensities and light deflection properties, the pixel values canvary significantly, depending on the ROI region drawn. This limitationmakes comparison between different experimental conditions difficult.Thus, we used the maximum RLU/min within a ROI, not integrated over thepixels, as the unit to compare the CCD signals, and the resultscorrelated very well with luminometer readings of tissue extracts (TableI) (90, 94).

Confocal Microscopy Study of Lung Metastasis

Mice with signal in the lungs were evaluated by confocal microscopy todetermine whether the signal from the reporter gene reflected metastaticevents. Animals were perfused with 2% paraformaldehyde, and lungs wereinflated with 3% agarose. Specimens were subsequently fixed by immersionin the same fixative, washed in PBS, and embedded on 7% agarose.Fragments from the tumor were used as positive control. Sections of 500μm were obtained with a vibrotome, and stained with human-specificanti-keratin antibody (AM273-5M, BioGenex, San Ramon, Calif.) and aCY-3-conjugated secondary antibody. Observation was performed on aconfocal microscope (BioRad 1024 confocal microscope, Hercules, Calif.),and ImagePro4.0 software was used for quantification of serial sections.

Immunohistochemical Analysis of Tumor Sections

Immunohistochemistry was performed according to Leav et al. (108).Briefly, tissue sections were deparaffinized, and antigen retrieval wasachieved by boiling in 0.1M sodium citrate pH 6.0 for 15 min. Tissuesections were incubated at 4 degrees C. overnight with respectiveantibodies, alpha-cytokeratin cocktail AM273-5M (BioGenex, San Ramon,Calif.), AR-beta 5 micro g/ml (UpState, Lake Placid, N.Y.), or PSA 1:40(Novocastra, Ontario L7N 3J5, Canada). Stringent blocking and washingprocedures were carried out to reduce background staining, prior toadding multilink 1:20 (BioGenex, San Ramon, Calif.) and AP label 1:20 atroom temperature for 20 min each. Finally, sections were washed againbefore color development with DAB (BioGenex, San Ramon, Calif.). Themicrographs were visualized with an Olympus BX41 microscope, and imagescaptured by an Olympus Camedia C-3030 digital camera. TABLE 1 Table 1.Summary of CCD image signal intensities and luminometry results ofluciferase expression in mice. Systemic Injections Naive days CMV/Animals a p.i. CMV (n) PSE-BC (n) PSE-BC b Max. CCD Imaging Signal(RLU/min) in liver 4 7.6E+7 7 402 7 1.9E+5 11 6.0E+7 3 952 3 6.3E+4Luminometry Readings (RLU/ug protein), liver 4 2.2E+7 5 70 5 3.1E+5Intra-tumoral injections Max. CCD Imaging Signal (RLU/min) in tumor (T)or liver (L) LAPC-4 days CMV CMV PSE-BC CMV/ (AD) p.i. (L) (n) (T) (n)(T) (n) PSE-BC b 4 3.3E+5 3 7.2E+4 3 897 5 8 1.1E+6 3 4.3E+4 3 1798 5 2411 c 4.2E+4 3 1309 4 15 1.0E+6 3 d 1237 5 21 6.7E+5 3 d 1311 4 LAPC-4days (AI) p.i. PSE-BC (n) AI/AD e 4 925 3 1 8 8586 4 4.8 11 12803 4 9.815 7498 4 6.1 21 3291 4 2.5a Non-tumor bearing, naïve mice.b Calculated by dividing average signals in the AdCMV-luc-treated groupby the respective signals in the AdPSE-BC-luc-treated group.c Incomplete data collected for this time point.d Unable to determine. Due to the dominant signals in the liver, thesignals in the tumors were below the minimum image analysis settingused.e Calculated by dividing the average signals for the AI LAPC-4 cohort bythe signals for the AD LAPC-4 cohort at the same time pointpost-injection.

EXAMPLE 4

The following example provides description of monitoring gene therapywith reporter gene imaging.

Advent of new techniques in molecular biology and their integration intonuclear medicine provides a great opportunity to improve the quality ofdiagnosis and treatment of many diseases. Methods are actively beingdeveloped for controlled gene delivery to various somatic tissues,including tumors, using novel formulations of DNA and for controllinggene expression using cell specific, replication-activated, anddrug-controlled expression systems (109-111). Although various methodsof gene therapy have met with very limited success, it is likely thateventually many disease processes will be successfully treated withdelivery of one or more genes to target tissue(s). A major concern forthe application of gene therapy is to achieve a controlled and effectivedelivery of genes to target cells and to avoid expression in non-targetlocations. Imaging the expression of a particular therapeutic gene islikely to be critical to optimizing gene therapy. Direct imaging of theexpression of every therapeutic gene would require development ofhundreds of different radiolabeled probes targeted against eachtherapeutic protein, and thus the development of a more general approachto indirectly monitor therapeutic gene expression is needed. Thereporter gene approach is one such potential approach that has been wellvalidated in pre-clinical models and is the focus of the current review.

Reporter Gene Concept

Reporter genes have long been used to study various aspects of geneexpression including, promoter/regulatory elements, inducible promotersand endogenous gene expression (112, 113). The general concept is thatregulatory regions of genes (e.g., promoters/enhancers) can be clonedand used to drive transcription (the process of converting DNA to mRNA)of a reporter gene. By introducing a reporter gene driven by a promoterof choice into target tissue(s), one can indirectly monitor expressionof the gene whose promoter has been cloned. This avoids having to builda specific probe to evaluate the expression of every new gene. The useof a promoter of choice also allows expression of the reporter gene onlyin select tissues, because specific promoters are active only inspecific tissues. Conventional methods used by molecular biologists tomonitor gene expression take advantage of reporter genes likeβ-galactosidase (114, 115) and chloramphenicol-acetyltransferase (CAT)(116, 117), which require tissue samples for determining theirexpression levels. Other methods make use of optical reporter genes suchas luciferase (118, 119), green fluorescent protein (GFP) (120-122) orβ-lactamase (123). The optical reporter genes have been used in livinganimal models as well but are not generalizable for human applications(124).

Due to technological innovations (125, 126) such as positron emissiontomography (PET) and single photon emission computed tomography (SPECT),it is now possible, using radiotracers (reporter probes), to imagereporter gene expression in vivo both repeatedly and non-invasively.Radiotracer imaging techniques offer the ability to monitor the detailedlocation, magnitude and time-variation of reporter gene expression.

Adapting the Reporter Gene Concept for Radionuclide Imaging

In order to adapt the reporter gene concept for imaging with PET orSPECT two primary approaches are possible (FIG. 18). The reporter genecan be chosen so that it encodes for an enzyme that is capable oftrapping a tracer by action of the enzyme on a chosen tracer. A secondapproach uses a reporter gene that encodes for an intracellular and/orextracellular receptor capable of binding a tracer (e.g., radioactiveligand). The accumulation of the tracer in both approaches is dependenton the expression of the reporter gene. Through the choice of the righttracer and optimization of it's pharmacokinetics it is possible toprimarily achieve imaging signal only in those areas in which thereporter gene is expressed. The introduced reporter gene driven by apromoter of choice is sometimes referred to as a transgene. Thisreporter gene-imaging paradigm is independent of a particular deliveryvector; it can be used with any of the several currently availablevectors (e.g., retrovirus, adenovirus, adeno-associated virus,lentivirus, liposomes, etc.). The common feature for all vectors is thecDNA expression cassette containing the reporter gene(s) of interest.The promoter can be constitutive, leading to continuous transcription,or can be inducible leading to controlled expression. The promoter canalso be cell specific, allowing expression of the reporter gene to berestricted to certain cells.

The ideal reporter gene would have the following characteristics: (A)When expressed, the reporter gene protein should produce specificreporter probe accumulation only in the cells in which it is expressed.(B) When the reporter gene is not expressed, there should be noaccumulation of the reporter probe in cells. (C) There should be noimmune response to the reporter gene product and this product should notsignificantly perturb the cell. Other desirable characteristics are alsoimportant and are reviewed elsewhere (127).

Specific Development of Radionuclide Reporter Genes

One of the earliest approaches investigated cytosine deaminase as areporter gene with 5-[3H]-fluorocytosine (5-FC) as the reporter probe(128). Cytosine Deaminase (CD), which is expressed in yeasts andbacteria but not in mammalian cells, converts the antifungal agent5-fluorocytosine to the highly toxic 5-fluorouracil (5-FU). The 5-FCdoes not incorporate into mammalian DNA synthesis pathway, however, itsby-product 5-FU can block DNA and protein synthesis due to substitutionof uracil by 5-FU in RNA and inhibition of thymidilate synthetase by5-fluorodeoxyuridine monophosphate, resulting in impaired DNAbiosynthesis. However, lack of sufficient accumulation of the probe incytosine deaminase expressing cells (due to efflux of 5-FU) limits itsuse as an imaging reporter gene (128). More recently a magneticresonance spectroscopy (MRS) based approach using some modeling has beendescribed using this reporter gene (129). The conversion of ¹⁹F-labeled5FC to 5FU was followed by MRS in subcutaneous human colorectalcarcinoma xenografts in nude mice by using H29 cell lines stablytransfected with yeast cd (ycd) gene and a three compartment model wasdescribed for non-invasive estimation of ycd transgene expression. Theutility of an MRS based approach warrants further investigation.

One of the most widely used reporter gene systems to image geneexpression is the herpes simplex virus type I thymidine kinase gene(HSV1-tk). Thymidine kinases are present in all mammalian cells; theyphosphorylate thymidine for incorporation into DNA. Unlike mammalianthymidine kinases, HSV1-TK has relaxed substrate specificity, and isable to phosphorylate acycloguanosine and uracil derivatives which get“trapped” in their phosphorylated state inside the cell. Two maincategories of substrates have been investigated as reporter probes forimaging HSV1-tk reporter gene expression: derivatives of uracil {e.g.,2′-fluoro-2′-deoxy-1-β-D-arabinofuranosyl-5-iodo-uracil (FIAU) labeledwith radioactive iodine} and derivatives of guanosine {e.g., penciclovir(PCV) radiolabeled with ¹⁸F}. These two major classes of reporter probesshare in common the ability to be phosphorylated by HSV1-TK, leading totheir accumulation in cell (130). Further details of the HSV1-tkreporter gene approach including a comparison of various tracers, aredetailed in a different review article (127). A mutant HSV1-TK enzyme(HSV1sr-39TK) that utilizes ganciclovir (GCV) and penciclovir substratesmore effectively, and thymidine less effectively than the wild-typeHSV1-TK enzyme, has been described and successfully applied in PETimaging (131). The HSV1-sr39tk mutant illustrates the ability toengineer the reporter protein and reporter probe to be optimized foreach other, leading to a marked gain in imaging signal.

Another radionuclide reporter gene system uses the dopamine type 2receptor (D₂R), which binds spiperone {(3-(2′[¹⁸F]-fluorethyl)spiperone(FESP)} intra- and extracellulary and thus results in probe accumulationin D₂R expressing cells/tissue (132, 133). The D₂R receptor is atransmembrane protein expressed predominantly in stratium and pituitaryand important for mediating the effects of dopamine to controlmovements. This receptor exerts its response through G-protein mediatedsignaling cascade involving adenyl cyclase as a second messenger.Spiperone, a D₂R antagonist can be labeled with ¹⁸F to yieldradiolabeled FESP and thus has been applied for PET imaging of the D₂Rreporter gene (133). Recently, we have investigated the potentiality oftwo mutant D₂R receptors (D₂R80A and D₂R194A) for PET imaging that canuncouple the downstream cAMP dependent signaling cascade while retainingthe property to bind with FESP (134). These two mutants have anadvantage of overcoming the undesirable effects of ectopic expression ofD₂R receptors on cellular biochemistry by uncoupling signaltransduction. The expression of HSV1sr-39tk and D₂R reporter genes canbe imaged simultaneously in living mice, with microPET, using twodifferent tracers (e.g., [¹⁸F]FPCV and [¹⁸F]FESP) respectively (FIG. 19)(135).

Somatostatin receptor subtype II (SSTr2) is another reporter gene thathas been extensively studied. Nuclear medicine physicians will befamiliar with the use of various somatostatin analogues (e.g.,radiolabeled octreotide) for imaging tumor cells expressing somatostatinreceptor. SSTR2 is normally expressed primarily in the pituitary gland,and also in other tissues like thyroid, pancreas, gastroienteric tract,kidney, lung etc. When SSTr2 is used as a reporter gene, the receptorcan be expressed on the surface of cells that would not normally expressthis gene, and therefore the use of various tracers can image SSTr2reporter gene expression. This approach is discussed in further detailin a review article by Rogers et. al. (136). Several other radionuclidebased reporter gene approaches have been preliminarily studied and arelisted with appropriate references in Table 2.

Gene Therapy Studies

Precise localization and quantitative assessment of the level of geneexpression is highly desirable for the evaluation of gene therapytrials. Most therapeutic transgenes lack appropriate ligands or probesthat can be radiolabeled and used to generate images that define themagnitude of therapeutic gene expression. Therefore it is usuallynecessary to develop and validate “indirect” imaging strategies using areporter gene in combination with a therapeutic gene. The goal of theseapproaches is to quantitatively image reporter gene expression and fromthat infer levels of therapeutic gene expression. Several approaches arecurrently being developed for indirect imaging of a therapeutic gene andare discussed next.

Fusion Approach

In recent years, fusion gene/protein technology has become a powerfultool in molecular biology, biochemistry, and gene therapy. A fusion geneconstruct contains two or more different genes joined in such a way thattheir coding sequences are in the same reading frame and thus a singleprotein with properties of both the original proteins is produced.Examples include HSV1-TK-GFP, (137-139) HSV1-TK-luciferase-Neo (140). Anadvantage of a fusion approach is that expression of both genes isabsolutely coupled. This approach, however, cannot be generalized, asmany fusion proteins do not yield functional activity for both theindividual proteins or may not localize in an appropriate sub-cellularcompartment. Every new therapeutic gene has to be fused to a reportergene, and often the reporter protein and/or therapeutic protein activityis partially compromised. This approach is therefore not asgeneralizable as the approaches discussed below.

Bi-Cistronic Approach

Another approach to express multiple genes from the same vectorconstruct is to insert an internal ribosomal entry sites (IRES) sequencebetween the two genes. Both the genes are transcribed into a single mRNAfrom the same promoter but are translated into two different proteinswith the help of the IRES sequences. The discovery of cap-independenttranslation and existence of internal ribosomal entry sites inpolioviruses and encephalomyocarditis virus opened a new gateway toconstruct bicistronic vectors for gene therapy purpose (141-143). IRESsequences are generally 450-800 bp long with complex RNA secondarystructures that are required for initiation of translation. Severalgeneral translation initiation factors like eIF3, eIF4A, eIF4G and somespecific cellular IRES transacting factors (ITAF's) are required todrive proper translation of the second cistron. We recently reported onesuch bi-cistronic vector where both D₂R and HSV1sr-39tk genes areco-expressed from a common promoter with the aid of EMCV IRES and imagedby microPET in multiple, stably transfected tumors in living mice. (FIG.20) (144). An approach using β-gal and HSV1-tk was also studied in micewith SPECT (145) Although IRES sequence leads to proper translation ofthe downstream cistron from a bicistronic vector, expression from theIRES could be cell type specific and the magnitude of expression of thegene placed distal to the IRES is often attenuated (144, 146). This canlead to a lower imaging sensitivity, and methods to improve thisapproach are currently under investigation. An IRES may also havedifferent behavior in different cell types; a property yet to be fullyexplored.

Double Promoter Approach

Two different genes expressed from distinct promoters within a singlevector (e.g., pCMV-D₂R-pCMV-HSV1-sr39tk) can potentially be a useful wayto couple the expression of two genes. This approach may avoid some ofthe attenuation and tissue variation problems of an IRES based approach,and is currently under active investigation.

Co-Vector Administration Approach

Another approach to express both the therapeutic and reporter gene canalso be achieved by co-administrating both the genes cloned in twodifferent vectors but driven by same type of promoter (147). Thisapproach has recently been tested and shows that the expression of twoPET reporter transgenes, HSV1-sr39tk and D2R, driven by same CMVpromoter but cloned in separate adenoviral vectors is well correlated atthe multi-cell (macroscopic) and tissue level when deliveredsimultaneously. However, it is important to realize that trans effectsbetween promoters on co-administered vectors carrying can potentiallyaffect reporter gene expression.

Bi-Directional Transcriptional Approach

For many gene therapy applications it would be desirable to not onlydeliver the therapeutic gene to desired target(s), but to be able toregulate levels of therapeutic gene expression. We have validated anapproach in a tumor model in living mice in which a simple antibiotic(doxycycline) can be used in combination with a fusion protein and abi-directional vector in order to correlatively express two genes ofchoice (148). This approach leads to bi-directional transcription, twomRNAs, and two proteins. It has the unique feature that the levels oftranscription can be regulated by levels of doxycycline. This approachavoids the attenuation and tissue variation problems of an IRES basedapproach and may prove to be one of the most robust approaches developedto date.

Non-Radionuclide Approaches to Reporter Gene Imaging

Radionuclide approaches offer a highly sensitive approach for imagingreporter gene expression that can easily be extended from animal studiesto human. Nevertheless, other modalities, when used in the appropriatesetting, may have desirable characteristics. Magnetic resonance imaging(MRI) techniques recently have obtained encouraging initial results.Using a substrate{(1-(2-(b-galactopyranosyloxy)propyl)-4,7,10-tris(carboxymethyl)1,4,7,10tetraazacyclododecane)gadolinium(III)-abbreviatedas EgadMe} that can be enzymatically processed by β-galactosidase(β-gal) and can generate MRI signal, Louie et al (149) recentlydemonstrated a MRI based in vivo assay of gene expression in livingXenopus laevis embryos. After injecting the β-gal mRNA in one of thecells of a two cell stage X. laevis embryo, reporter gene expression wasfollowed by introducing EgadMe. The MR images showed high signalintensity in the embryo with both β-gal mRNA and EgadMe in contrast tothe embryo with EgadMe alone. Following image correction, it is possibleto recognize the eye and branchial arches. The MR image of a live embryocorrelates well with images of the same embryo after fixation andstaining with X-gal. This approach for routine in vivo use is currentlylimited due to the delivery of the EgadMe substrate which does not havedirect cellular uptake. In another study, Weissleder et al described thefirst non-invasive in vivo MR imaging of transgene expression (150).They implanted nude mice with 9 L gliosarcoma cells stably transfectedhuman transferrin receptor (HETR) and control transfected (ETR−) cellsfollowed by injection of iron oxide nanoparticles conjugated with humanholo-transferrin (Tf-MION) to image the tumors with MRI. Other MRI/MRSbased approaches are reviewed elsewhere (151).

Optical reporter genes such as firefly luciferase or green fluorescentprotein (GFP) are currently being used to monitor gene expression invivo (124, 152). Firefly luciferase gene expression can be imaged usinga high sensitivity charged coupled device (CCD) camera. Light isproduced through the interaction of luciferase with its substrateluciferin (injected peritoneally) in presence of Magnesium and ATP(152). For the green-fluorescent protein (GFP), an input wavelength mustbe provided and an output wavelength of light is produced which can beimaged optically by a florescence microscope (153). These opticalapproaches have the distinct advantage of low background signal, ease ofuse, and low cost; however, they are limited by light scatter andabsorption, limiting studies in deep tissues. Although these opticalreporter genes will be extensively used for basic research in smallanimals, they are not generalizable for human applications. Furtherexamples are referenced in Table 2.

Human Imaging Studies for Gene Therapy

Studies to image reporter gene expression in human subjects are nowbeginning. Yaghoubi et. al. (154) reported a study to measure thekinetics, biodistribution, stability, dosimetry and safety of [¹⁸F]FHBGin healthy human volunteers, prior to imaging patients undergoingHSV1-tk gene therapy. The study by Yaghoubi et. al. indicated that dueto the properties of stability, rapid blood clearance, low backgroundsignal, biosafety and acceptable dosimetry [¹⁸F]FHBG should be a goodreporter probe for HSV1-tk imaging in humans, although [¹⁸F]FHBG doesnot significantly cross the blood-brain-barrier. This may eventuallyallow not only imaging of gene therapy performed with HSV1-tk, but ofany therapeutic gene coupled to HSV1-tk for indirect imaging of thetherapeutic gene. In another study, Jacobs et al (155) have shown that[¹²⁴I]FIAU, another PET reporter probe for HSV1-tk cannot penetrate theblood brain barrier and thus is not a good marker probe for noninvasivelocalization of HSV1-tk in the central nervous system. However, thisprobe is useful for areas where the blood brain barrier is disrupted(e.g., glioblastoma). The other reporter genes also have various tracersthat have been previously used in human subjects, and therefore canlikely be translated to human applications in gene therapy. With therapid progress in human gene therapy and molecular imaging it is likelythat Nuclear Medicine will play a major role in optimizing gene therapy.

Other Applications of Reporter Genes

Animal applications of the reporter gene imaging assays are particularlyimportant because they allow for the study of many important biologicalissues in living animals. The use of transgenic animals carryingreporter genes offers unique possibilities for tracking a single animalrepeatedly over time during experimental manipulations. Transgenicanimals expressing the HSV1-tk gene from tissue specific promoters havebeen developed to perform cell specific ablation followingadministration of pharmacologic levels of pro-drugs such as ganciclovir.A transgenic mouse model in which the HSV1-tk marker/reporter gene isdriven by the albumin promoter has been studied (156). Thealbumin-HSV1-tk transgenic mice have been imaged on a microPET with both[¹⁸F]FPCV and [¹⁸F]FHBG and clearly demonstrate accumulation ofmarker/reporter probe in the mouse liver at one hour after injection.Restriction of reporter probe accumulation in the liver is the result oftissue specific transcriptional activation of the HSV1-tkmarker/reporter gene by the albumin promoter. The albumin-HSV1-tk micewill be very useful for comparing alternate substrates in vivo and forassessing the reproducibility of assays. Furthermore, these transgenicmice will allow the study of albumin regulation under variousmanipulations (157). It would be very difficult to develop a tracerspecifically targeting albumin, and therefore this indirect approach ofimaging albumin gene expression (by imaging HSV1-tk reporter geneexpression) illustrates the power of a reporter gene approach to studyregulation of an endogenous promoter.

Other applications of reporter genes include: (1) imaging of celltrafficking by marking specific subsets of cells with a reporter gene exvivo or through transgenic models. (2) optimization of gene delivery inanimal models by studying various vectors carrying a reporter gene. (3)studies of the interaction of tumor cells and the immune system bymarking each with a distinct reporter gene. (4) studies of viralinfections by marking the virus of interest with a reporter gene. (5)studies of basic cancer biology by monitoring specific gene expressionin various tumor models.

Conclusion

Molecular and functional imaging allows the clinician/researcher tovisualize the cellular and/or molecular processes in living tissues. Thedevelopment of molecular imaging probes is the key to the importance andgrowth of nuclear medicine. Reporter genes have emerged as a verypowerful tool to monitor the delivery, magnitude, and time-variation oftherapeutic gene transfer in vivo. The radionuclide based reporter geneslike HSV1-tk, D₂R, SSTr2, etc., are currently being used for PET, SPECTand gamma camera imaging. Though still in their infancy, thenon-radionuclide based reporter genes are also emerging as another toolfor three dimensional, real time, noninvasive imaging of cellular andmolecular processes. Nuclear Medicine should help to lead the way formolecular imaging with reporter genes and probes for use with smallanimal and human imaging. TABLE 2 Summary of Reporter gene/probe systemsReporter gene Mechanism Imaging agents Imaging References CytosineDeamination [³H]-5- Cell Culture 128 deaminase fluorocytosine study[¹⁹F]-5 MRS 129 fluorocytosine Herpes-simplex Phosphorylation[¹³¹I]FIAU, SPECT, gamma 158 virus type 1 [¹⁴C]FIAU camera thymidinekinase [¹³¹I]FIAU SPECT, gamma 159 (HSV1-tk) camera [¹²⁴I]FIAU PET 160[^(123/125)I]FIAU Gamma camera 161 [¹²³I]IVDU, Cell Culture 162[¹²⁵I]IVFRU, [¹²⁵I]IVFAU, [¹²⁵I]IVAU [¹²⁵I]FIAU, Cell Culture 163[¹²⁵I]FIRU [³H]FFUdR Cell Culture 164 [¹⁴C]GCV, Autoradiography 165,166, 167 [³H]GCV [¹⁸F]GCV PET 167, 168 [¹⁸F]PCV PET 135 [¹⁸F]FHPG PET169, 170, 171, 172, 173 [¹⁸F]FHBG PET 174, 147 Mutant Herpes-Phosphorylation [¹⁸F]PCV Cell culture, PET 131, 144, 147 simplex virus[¹⁸F]FHBG PET 148 type 1 thymidine kinase (HSV1- sr39-tk) Dopamine2Receptor-ligand [¹⁸F]FESP PET 133, 144, 147, 148 receptor MutantReceptor-ligand [¹⁸F]FESP PET 134 Dopamine2 receptor SomatostatinAffinity binding [¹¹¹In]DTPA-D- Gamma camera 175 receptorPhe¹-octreotide [⁶⁴Cu]-TETA- Tumor uptake 176 octreotide study^([188)Re]- Gamma camera 177, 136 somatostatin analogue, ^(99 m)Tcsomatostatin analogue Oxotechnetate- Binding via [^(99 m)Tc]Autoradiography, 151 binding fusion transchelation Oxotechnetate Gammacamera 178 proteins Gastrin releasing Affinity binding [¹²⁵I]-mIP-Des-Cell culture 179, 180 peptide receptor Met¹⁴-bombesin(7-13)NH_(2,)[¹²⁵I]bombesin, Cell Culture 181 [^(99 m)Tc]-bombesin Cell Culture 182analogue Sodium/Iodine Active symport [¹³¹I] Gamma camera 183, 184symporter (NIS) Tyrosinase Metal binding to Synthetic Cell culture/MRI185 melanin metallomelanins [¹¹¹In], Fe 186 Green Fluorescent GFP geneFluorescence fluorescence 187, 153, 188, protein (GFP) expressionmicroscopy 189, 190, 191, 192 resulting in fluorescence Luciferase(firefly) Luciferase - Bioluminescence Charge coupled 152, 193 luciferinreaction device (CCD) in presence of Mg²⁺ camera Cathepsin D QuenchedNIRF Fluorescence CCD camera 194, 195, 196 fluorochromes activationβ-galactosidase Hydrolysis of β- {(1-(2-(b- MRI 149 glycoside bondgalactopyranosyloxy)propyl)- 4,7,10-tris(carboxymethyl)1,4,7,10tetra-azacyclododecane)gadolinium(III) or EgadMe Engineered Receptor-ligand,Superparamagnetic MRI 150 transferrin internalization iron Tf-MIONOxide-tran receptor (TfR)

EXAMPLE 5

The androgen-dependent (AD) phase of prostate cancer requires afunctional androgen receptor (AR) and physiological levels of its liganddihydroxytestosterone (DHT). Prostate cancer eventually transitions toandrogen-independent (AI) growth after androgen withdrawal (i.e.,castration). A major issue is whether AR is functional in AI cancer. Weemployed gene-expression based molecular imaging and chromatinimmunoprecipitation (ChIP) to study AR dynamics during cancerprogression in SCID mice bearing human prostate cancer xenografts. Acharge-coupled device optical imaging system and an adenovirus-based,prostate-specific imaging vector were used to visualize the loss of ARactivity after castration and restoration of activity upon transition toAI growth. The imaging signal in AI cancer correlated with twobenchmarks of AR function in AD cancer: nuclear localization of AR andrising prostate specific antigen (PSA) levels. Chromatinimmunoprecipitation (ChIP) data suggest that AR is initially bound tothe endogenous PSA enhancer/promoter in AD tumors, releases from the DNAupon castration, but rebinds in the AI state. RNA polymerase II does notdissociate from the PSA gene after castration but its preferreddistribution changes from coding regions to the promoter, potentiallyfacilitating the AI transition. Our study demonstrates that AR isfunctional in and therefore likely to facilitate the AI phase ofprostate cancer.

Prostate cancer growth is initially dependent upon the androgen receptor(AR) (Gelmann 2002), a member of the steroid receptor subfamily ofnuclear receptors (Freedman 1999). In the presence of its ligand,dihydroxytestosterone (DHT), AR moves from the cytoplasm to nucleus,binds to 15-bp DNA elements (AREs) in enhancers or promoters, andactivates expression of genes involved in prostate metabolism. Prostatecancer initially ceases growth with treatments that lower theconcentration or effectiveness of DHT. The cancer eventually progressesfrom this androgen-dependent (AD) state to an androgen-independent (AI)state upon failure of androgen blockade therapies (Abate-Shen and Shen2000; Feldman and Feldman 2001; Arnold and Isaacs 2002). AR is known tobe expressed, and sometimes overexpressed, in many AI cancers (Visakorpiet al. 1995; Gregory et al. 2001) but it has not been shown to befunctional. The functionality is an important issue. If ARtranscriptional activity is resuscitated in the ligand-deprivedenvironment it would provide a rationale for progression of AI cancer.

New molecular imaging technologies have made it possible tonon-invasively visualize gene activity in real time in living subjects(Contag et al. 2000; Herschman et al. 2000; Contag 2002; Massoud andGambhir 2003). The development of gene expression- and vector-basedimaging approaches permits repetitive and quantitative measurements ofgene regulation in a spatial and temporal fashion. These technologiesinclude optical imaging with cooled charge coupled devices (CCD)(O'Connell-Rodwell et al. 2002) and radionuclide approaches such aspositron emission tomography (PET) and single photon emission-computedtomography (SPECT) (Gambhir 2002). The pioneering studies in this areaoriginally used potent but constitutive regulatory elements to drivelevels of reporter gene expression that could be detected by CCD orradionuclide technologies.

Our goal was to monitor AR-mediated transcription during the progressionof prostate cancer to the AI state in living animals. We employed CCDoptical imaging of firefly luciferase because the short half-life ofluciferase in conjunction with highly active reporter genes facilitatesdynamic measurement of expression occurring over weeks in living animals(Wu et al. 2001a). A major challenge in creating such a system was togenerate a robust, prostate-specific, reporter cassette that coulddetect optical signals in a tumor throughout the course of cancer andrespond to changes in AR activity. A second challenge was to correlatethe transcriptional activity with benchmarks such as AR nuclearlocalization and binding of AR to endogenous promoters. The correlationwould ensure that the optical signals were accurately reportingmolecular events. We focused our efforts on the prostate specificantigen (PSA) gene because of its AR-responsiveness and prostatespecificity (Cleutjens et al. 1996b).

PSA is a secreted kallikrein protease widely used for evaluatingtreatment and progression of cancer although it has some drawbacks inprognostic utility (Bok and Small 2002). The PSA promoter and enhancerhave been extensively delineated. Both the promoter and enhancer containAREs necessary for transcriptional activity in AD cancer cell lines likeLNCaP (Pang et al. 1995; Cleutjens et al. 1996a; Schuur et al. 1996;Cleutjens et al. 1997a; Cleutjens et al. 1997b; Pang et al. 1997; Zhanget al. 1997). A 440-bp core segment of the enhancer plays the major rolein androgen-responsiveness (Cleutjens et al. 1997b). This core segmentcontains a cluster of AREs, which bind AR cooperatively and contributesynergistically to gene expression (Huang et al. 1999; Reid et al.2001).

We previously exploited the synergistic nature of AR action to augmentthe AR-responsive activity of the PSA enhancer (Wu et al. 2001b). Oureffort was designed to track AR activity in cancer via molecularimaging. We re-engineered the PSA regulatory region by duplicating thecore portion of the PSA enhancer and by removing non-essential DNAbetween the enhancer and proximal promoter. This strategy generated achimeric construct, termed PBC, which maintained low basal expression innon-prostate tissues but enhanced prostate-specific, AR-responsiveactivity by 20 fold.

The enhanced activity and specificity of the chimeric constructs wereretained in an adenoviral vector expressing firefly luciferase. Theadenovector bearing this “first generation” imaging cassette, AdPBC, wasable to detect distal metastatic lesions in SCID mouse xenograft modelsupon intratumoral or systemic injection via tail veins, andvisualization with a cooled CCD optical imaging system (Adams et al.2002). Although the duplicated enhancer provided a significant gain inactivity versus the original PSA enhancer, the overall activity was onlyabout 1-5% that of CMV in side-by-side comparisons. This low level ofactivity made it difficult to dynamically monitor the androgen-responsedue to the extended time frames required to observe a firefly luciferasesignal.

To further improve the activity we employed a strategy termed two-steptranscriptional activation (TSTA). The concept of TSTA was based on anapproach originally used to identify enhancers in drosophila (Brand andPerrimon 1993). In our TSTA system, the PSA regulatory region was usedto express the potent artificial transcription activator GAL4-VP16.GAL4-VP16 acts on a GAL4-responsive firefly luciferase reporter gene,increasing expression well beyond that observed with reporter constructsbearing the PSA regulatory region alone (Iyer et al. 2001). We optimizedthe system by using GAL4-derivatives containing one, two and four VP16(VP1, 2 and 4) activation domains, which acted on reporters containingone, two, five and nine GAL4 sites (G1, 2, 5 and 9) (Zhang et al. 2002).Our analysis indicated that the optimal system comprised the augmentedPSA enhancer, PBC, driving GAL4-VP2 on a G5 promoter. This approachresulted in robust amplification of expression in cell culture, whilemaintaining prostate and ligand response. The optimal system displayedactivities significantly higher than the CMV enhancer-driven fireflyluciferase (Iyer et al. 2001; Zhang et al. 2002).

In this paper we incorporated the optimal TSTA system into a replicationdeficient adenovirus using the simplified recombination system describedby Vogelstein and colleagues (He et al. 1998). We chose adenovirus as avector because it has high infection efficiency and is widely used ingene transfer studies into animals and humans (Pfeifer and Verma 2001).We injected the virus into AD and AI tumors implanted into SCID mice. Wedemonstrate the ability to image activation, inactivation andreactivation of AR activity during cancer progression. We coupled theimaging with immunohistochemical staining and chromatinimmunoprecipitation of AR on the PSA regulatory region in the variousstages of tumorigenesis. We generate strong support for the concept thatAR is fully active in AI cancer. Our ChIP data also suggest a modelwhereby the transcription complexes on AR-responsive genes do notdisappear in the absence of androgen but remain marginally active andpoised to resume full activity in the AI state.

Materials and Methods

Adenovirus Constructs

AdTSTA was generated from the optimal TSTA plasmid (Zhang et al, 2002).A second NotI site 5′ from the PBC enhancer was removed to create uniqueNotI site in the vector. A SalI-NotI fragment containing the coreBCVP2G5-Luc fragment was excised by NotI and partial SalI digestion andinserted into the SalI-NotI site of pShuttle vector (Q-Biogene,Carlsbad, Calif.), which was then incorporated into the adenovirusvector AdEasy™ through homologous recombination. AdCMV was generated asprevious described (Adams et al. 2002). The viruses were packaged andpropagated in 293A cells (293 cell line stably expressing the E1A gene).The virus was scaled up, purified via a CsCl gradient and titered byplaque assays on 293 monolayers (infectious units=plaque-forming units).Virus is stored at ˜1011 pfu/ml in 10 mM Tris-HCl, 1 mM MgCl₂, and 10%glycerol.

Cell Culture and Xenografts

The human prostate cancer cell line LNCaP was grown in RPMI 1640supplemented with 10% Fetal Bovine Serum and 1% Penicillin/Streptomycinsolution. HeLa, MCF-7, and HepG cells were cultured in DMEM with 10%Fetal Bovine Serum and 1% Penicillin/Streptomycin. Prior totransfection, cells were transferred for 24 hours into medium containing5% charcoal stripped serum (Omega Sci. Tarzana Calif.). The syntheticandrogen Methylenetrienolone (R1881; NEN Life Science Products, Boston,Mass.) was added to “ligand positive” samples where indicated.

Human prostate tumor xenografts were generated on SCID mice aspreviously described (Klein et al. 1997). Briefly, approximately 1×10⁶LAPC-9 tumor cells generously provided by Dr. Charles Sawyers were mixed1:1 with Matrigel (Collaborative Research, Bedford, Mass.) and implantedsubcutaneously on the left flank of male SCID (scid/scid) mice. The AIsublines were passaged several rounds in castrated male mice.Single-cell suspension cultures were maintained on PreBM/GM media(Clonetics, Walkersville Md.). Alternatively, tumors were extracted fromfounder mice, minced into ˜0.2 mm cubes, bathed in matrigel, andimplanted subcutaneously onto the left flanks of SCID mice.

Virus Activity Assays

For luciferase assays, the cultured cells were infected with AdTSTA orAdCMV at an MOI of 0.1. After 48 hours, the cells were harvested andlysed using the passive lysis buffer provided in the firefly luciferaseassay kit (Dual-Reporter Luciferase Assay System, Promega, Madison,Wis.). Firefly luciferase activities of 5% of the cell lysatessupplemented with 100 μl of D-luciferin were measured using aluminometer (Lumat 9507, Berthod Germany) with an integration time of 10sec.

Immunoblot Analysis of GAL4-VP16 Expression

LNCaP cells were grown in 10-cm dishes and infected with AdTSTA at MOI10. Forty-eight hours later the cells were harvested and lysed with RIPAbuffer (10 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 1% DOC, 1 mM EDTA and 1%NP40). Protein concentrations of the extracts were normalized (Bio-RadDc protein assay Kit), the samples were fractionated on 4-15% gradientacrylamide gels (Bio-Rad, Hercules, Calif.) and subjected to immunoblotanalysis with rabbit polyclonal antibodies generated against intactGAL4-VP16 or loading control proteins.

CCD Imaging to Detect In Vivo Luciferase Expression

For the naïve mice, 107 pfu of AdTSTA or AdCMV suspended in 100 μlphosphate buffered saline (PBS) was injected via the tail vein. For theLAPC9 xenografts, a total of 107 pfu of AdTSTA or AdCMV in 40 μl PBS wasinjected directly into the 0.5-cm diameter tumor xenografts at multiplelocations. To ensure adequate distribution throughout the tumor, theinjection was carried out twice on two sequential days. The virus wasallowed to express the encoded genes and distribute throughout thetissue for 3-4 days prior to imaging. At the days specified in thefigures, the mice were anesthesized and injected with ˜150 mg/kgD-Luciferin (approximately 3 mg/mouse). Light signals (CCD images) wereobtained using a cooled IVIS CCD camera (Xenogen, Alameda, Calif.) andimages were analyzed with IGOR-PRO Living Image Software, whichgenerates a pseudoimage with an adjustable color scale. We determinedthe maximum photons/second of acquisition/cm2 pixel/steridian (sr)within a region of interest to be the most consistent measure forcomparative analysis. The imaging results correlated closely withluminometry of tissue extracts. Typically our acquisition times rangedfrom 1 to 10 seconds. These acquisition times were valid in tumorsinjected only 2 days prior to imaging.

Tumor Immunohistochemical Analysis

Immunohistochemistry was performed on paraffin-embedded tumor sectionswith antigen retrieval. Tissue sections were incubated at 4° C.overnight with anti-AR 5 μg/ml (UpState, Charlottesville, Va.). Afterstringent blocking, washing and incubation with multi-link (BioGenex,San Ramon, Calif.) and alkaline phosphatase label for 20 min at roomtemperature, sections were washed and developed according to themanufacturer's instructions (BioGenex).

Tumor Chromatin Immunoprecipitation

Tumors were extracted from the mice and washed with ice cold PBS. Thetumors were quickly minced and immersed in 1% formaldehyde solution,where they were further minced and homogenized using a glass dounce. Thetotal incubation in formaldehyde solution was for 30 minutes. Prior tosonication, the cell suspensions were washed 10 minutes each in solutionI containing 0.25% Triton, 10 mM EDTA, 1 mM EGTA and 10 mM HEPES, pH7.5, and in solution II containing 0.2 M NaCl, 1 mM EDTA, 1 mM EGTA and10 mM HEPES, pH 7.5. Extracts were obtained by 8×15 sec sonication inlysis buffer containing 1% SDS and 10 mM EDTA using a Fisher ScientificModel 550 sonicator at setting 4 with a microtip. Chromatin was purifiedfrom insoluble debris by micro-centrifugation at 15,000 rpm for 20 min.

To perform immunoprecipitation, the chromatin was diluted 1:7 indilution buffer containing 70 mM HEPES, pH 7.5, 2.5 mM NaCl, 1.5 mMEDTA, 1.5% Triton and 0.6% deoxycholate. The extracts were pre-clearedwith pre-immune IgG together with Sepharose A or G agarose beads(Amersham, Piscataway, N.J.) for 1 hour at 4° C. Pre-cleared extractswere incubated with 4 to 6 μg of specific antibodies at 4° C. overnightfollowed by incubation with 30 μl of agarose A or G beads for 1 hour thenext day. The antibodies included the N20 and C19 AR antibodies fromSanta Cruz, Biotech; the pol II CTD8WG16 monoclonal from QED Bioscience(San Diego, Calif.); and a TFIIB antibody generated in our lab. Thebeads were washed twice with buffer containing 50 mM HEPES, pH 7.5, 0.15M NaCl, 1 mM EDTA, 1% Triton, 0.5% deoxycholate and 0.15% SDS followedby wash with LNDET buffer containing 0.25 M LiCl, 1% NP40, 1% DOC, 1 mMEDTA and 10 mM Tris, pH 8.0. The protein-DNA complexes were then elutedfrom the beads with 30 to 50 μl elution buffer containing 1% SDS and 1mM NaHCO3. The eluates were diluted with TE and incubated at 65° C.overnight to reverse the cross-link. The samples were then treated withproteinase K at 100 ng/μl and RNAase A for 1 h at 55° C. and thenextracted with phenol. The DNA was then precipitated with ethanol,resuspended in 30 μl water and subjected to PCR analysis. Typically one1 cm tumor yielded enough material for 6 PCR reactions. The onput samplein the data shown in the ChIP experiments was typically 2% of the DNAadded to a ChIP reaction.

The PCR analyses were performed with four sets of 32P-labeled ofprimers: Enhancer: 5′GGTGACCAGAGCAGTCTAGGTG3′ and5′TGTTTACTGTCAAGGACAATCGAT3′ Promoter: 5′GTATGAAGAATCGGGGATCGT3′ and5′GCTCATGGAGACTTCATCTAG3′ Middle: 5′TATGCTTGGGGACACCGGAT3′ and5′TTAGAGCTGGAGTGGAAGGATAT3′ Exon 5:5′TAATGGTGTGCTTCAAGGTATCACG3′ and5′GTGTCCTTGATCCACTTCCGGTAAT3′

The PCR cycling protocol was 40 sec at 94° C., 3 minutes at 75° C., 2minutes at 65° C., followed by 20 cycles of 40 sec at 94° C., 1 minuteat 65° C. and 2 minutes at 72° C., followed by a 10 minute extension at72° C. The PCR products were phenol-extracted, separated on 4%polyacrylamide gels and autoradiographed by exposure to XAR-5 film.

Results

The Adenovector TSTA Imaging System

FIG. 22 illustrates the adenoviral-based TSTA cassette employed in ourstudy. A modified chimeric PSA promoter containing two copies of theAR-responsive core PSA enhancer is used to drive expression of thefusion protein GAL4-VP2, bearing two copies of the VP16 activationdomain. This strategy ensures robust, AR-mediated, prostate-specificexpression of GAL4-VP2. In the second step, GAL4-VP2 binds to five GAL4binding sites positioned upstream of the adenovirus E4 core promoter andactivates high levels of firefly luciferase in prostate-derived tissues.Luciferase is measured in cell culture by luminometry and in vivo inD-Luciferin-injected live animals using a cooled CCD optical imagingsystem (Wu et al. 2001a). The two expression cassettes were joined in adivergently-expressed orientation in the genome of Adenovirus serotype5, with the E1 and E3 coding regions deleted, which renders the packagedvirus replication deficient (He et al. 1998). To validate the ability ofthis imaging system to accurately detect AR activity we tested itsactivity, cell specificity and response to androgen in cell culture andthen addressed the same criteria in SCID mice with human prostate cancerxenografts. Finally, we correlated the imaging activity with serum PSAlevels, AR nuclear localization and binding of AR to the endogenous PSAgene in tumors harvested from the SCID mice.

Prostate Cancer Specificity of AdTSTA

To assess the expression and androgen responsiveness of the AdTSTAsystem in cell culture, we infected the model androgen dependentprostate cancer cell line, LNCaP, with AdTSTA (FIG. 23A). Expression ofGALA-VP2, and firefly luciferase were activated significantly in thepresence of the synthetic androgen agonist R1881. The largestfold-increase of luciferase activity was observed at the 48-hour timepoint (FIG. 23A bottom), which correlated with the appearance ofGAL4-VP2 by immunoblot analysis (FIG. 23A, top).

The AdTSTA system maintained cell selectivity in culture (FIG. 23B). Anexample of these findings is shown for cells derived from prostatecancer (LNCaP), liver (HepG) and cervical cancer (HeLa) (FIG. 23). LNCaPis an androgen-dependent prostate cancer cell line (Horoszewicz et al.1983), which contains AR and secretes PSA (Tilley et al. 1990;Montgomery et al. 1992). AR expression is not observed in HeLa and HepG.PCR analysis demonstrated that viral infectivity was similar within atwo-fold range among the cell lines we tested. The LNCaP cells displayedsignificantly higher firefly luciferase activity than HeLa and HepG andresponded well to the androgen agonist R1881. MCF-7 cells, an ARexpressing breast cancer cell line was also tested but displayed only alow basal level of TSTA expression in the presence of R1881. Thus, theAdTSTA system responds to AR specifically in prostate cells.

High levels of luciferase are necessary to observe signals fromcell-specific promoters. Previous engineering of the PSA enhancer led tothe adenovector, AdPBC, which expressed firefly luciferase in a prostatespecific manner but with only 5% the activity of CMV. The low level ofactivity was suitable for initial in vivo studies but two problemslimited its utility in evaluating AR function in real time: The extendedtime frame necessary to observe firefly luciferase expression in tumorsand the long acquisition times on the CCD optical imaging system.Side-by-side comparison (FIG. 23B) demonstrated that AdTSTA displayed aligand induced firefly luciferase activity 10-fold greater than AdCMV(FIG. 23B) and nearly 200-fold greater than AdPBC. This gain of activityreduced the time frame required to observe a robust CCD signal from aweek to two days, and reduced average CCD acquisition times on averagefrom 5 minutes to a few seconds, thereby permitting us to perform theanalysis described below.

The specificity was then evaluated in vivo by tail vein injection intonon-tumor bearing SCID mice. FIG. 24 shows that tail vein injection ofAdCMV leads to significant expression in the liver of SCID mice lackingtumors. The figure presents CCD imaging data from a representative mouseat day 14. AdCMV generated a strong liver signal (˜106photons/sec/cm2/sr) on day 6, the signal peaked at day 14 (107photons/sec/cm2/sr) and then decayed slightly at day 22, when theexperiment was terminated. The liver signal was confirmed by imaging theindividual organs after sacrifice. In contrast to the data with AdCMV,injection with the same dose of AdTSTA did not result in detectableliver signals in male mice lacking tumors.

The AdCMV and AdTSTA viruses were then injected intratumorally into micebearing the LAPC9 xenografts (FIG. 24B). LAPC9 is a human prostatecancer derived from a bone metastasis, which was then implanted intoSCID mice (Klein et al. 1997). The tumor expresses PSA and wild-type AR.LAPC9 tumors respond to androgen in vivo. Upon castration of male mice,the tumor transiently halts growth and gradually transitions into the AIstate. Continued passage of the AI tumors in castrate mice generates astable AI model, which expresses AR and PSA. We employed an adenovectorbecause of the flexibility it offered, i.e., testing a wide variety oftumors, and because of the difficulty in establishing xenografts thatexpress the TSTA reporter system in a stable manner.

Direct intratumoral injection of the viruses into LAPC9 AD xenograftsfollowed by CCD imaging demonstrate that the AdTSTA is routinely twiceas active as AdCMV in the tumors (FIG. 24B). Typically, injection of 10⁷pfu of AdTSTA generated a robust signal. Therefore, we were able toreduce the amount of virus versus the 109 pfu employed in our previousimaging study with AdPBC (Adams et al. 2002).

Virus leakage of AdCMV resulted in signals emanating from the liver. Theliver signal was confirmed after sacrifice by imaging the individualorgans. In contrast, the specificity of the AdTSTA system, whose tissuedistribution parallels that of AdCMV, precluded liver expression.

We note that the AdTSTA-injected mice did not display a signal in theprostate either by tail vein or intratumoral injection. Under thecurrent conditions we are not achieving substantial delivery to theprostate probably due to virus sequestration in liver and lung (Wood etal. 1999). We did not pursue this issue because our goal was to study ARactivity in the xenograft. However, we are currently attempting todiscern the specific injection routes and protocols necessary to obtainprostate infection.

AR Signaling is Active in AI Tumors

To demonstrate the androgen responsiveness of the tumors we firstcastrated male mice bearing LAPC9 AD tumors and measured serum PSAlevels (FIG. 25). A decrease or plateau of serum PSA is indicative ofsuccessful hormone blockade therapy in humans. The xenograft datarevealed that the PSA levels ceased rising and dropped slightlybeginning on day 1 and remained flat for many days post-castration,recapitulating the clinical response of androgen blockade therapy. Atlater time points the PSA began to rise as tumors transitioned from ADto AI, an issue that is discussed further in a later figure.

Analysis of AD and castrated AD (ADc) tumors revealed that the imagingsignal responded well to castration (FIG. 26, center panels). TheAdTSTA-injected tumors typically emitted >107 photons/sec/cm2/sr on day4 after virus injection. Castration on day 4 led to a >10-fold drop inthe imaging signal by day 10 (FIG. 26 left panels: AD vs. ADc, p=0.01).In contrast, AdTSTA displayed robust activity in established AI mice.Interestingly, the signal actually increased from day 4 to day 10 in AI,whereas no significant increase was observed in AD over the same timeframe. These data indicate that the AR-responsive TSTA system isspecifically responding to the loss of AR activity in the ADc tumor butthat activity is regained in established AI tumors. We will show belowthat this loss and gain of activity correlates with the drop and rise ofPSA in ADc and AI tumors, respectively.

The imaging activity correlates well with the subcellular locale of AR(FIG. 26 right panels). AR is known to localize predominantly to thenucleus in the presence of androgen. In the absence of androgen, ARbecomes more diffuse and localizes to both the cytoplasm and nucleus(Gregory et al. 2001). Immunohistochemical analyses of tumors from thesacrificed LAPC9 animals showed that AR was tightly localized to thenucleus of AD tumors, became diffuse in ADc, but returned primarily tothe nucleus in established AI tumors. The staining in ADc washeterogeneous with a small number of cells showing nuclear staining butmost showing a diffuse pattern of nuclear and cytoplasmic staining.

Taken together the imaging and cytology data suggest that AR has resumedfunctioning in AI cancer. However, numerous other proposal have beenmade to explain how androgen-responsive genes might function in cellslacking physiological levels of androgen. For example, NF-κB has beenproposed to bind regulatory regions of androgen-responsive genes andmight substitute for AR in AI cancer (Chen and Sawyers 2002). However,the predominant nuclear location of AR in AI tumors suggest AR may befunctional. One prediction of this hypothesis is that AR should be bounddirectly to responsive enhancers and promoters in AI cancer.

Transcription Complex Assembly in Tumors Correlates with the ImagingSignal

To address our hypothesis we measured AR binding to the endogenous PSAregulatory region using chromatin immunoprecipitation (FIG. 27). The PSAenhancer and promoter have been well delineated. Previous chromatinimmunoprecipitation experiments performed in cell culture have revealedAR and RNA polymerase II (pol II) binding to the enhancer and promoterin LNCaP cells (Kang et al. 2002; Shang et al. 2002). We confirmed theseresults in LNCaP cells and then used that knowledge to analyze AR andpol II binding to the PSA gene in the context of AD and AI tumor samplesderived from LAPC9 tumors. The tumor ChIP experiments were significantlymore difficult to perform than the cell culture ones because the tumoris highly heterogeneous, solid tissue. We rapidly minced and crosslinkedthe tissue during tumor harvest and performed sonication in detergentcontaining high levels of SDS followed by ChIP in the presence of lowSDS and high levels of deoxycholate.

We analyzed AR binding to four regions of the gene: The enhancer, thepromoter, a region located between the enhancer and promoter, anddownstream exon 5 (FIG. 27). We predicted that AR would bind only to theenhancer and promoter. FIG. 27 demonstrates that our procedure workedreasonably well and AR binding was detectable above background to theenhancer and promoter in both the AD and AI samples, although it wasmore evident in AD in the experiment shown. Each experiment wasperformed three times and specific signals were confirmed by comparingthe AR-antibody signal to the background observed in a mockimmunoprecipitation with IgG. The promoter signals were lessreproducible than those on the enhancer region (see graph). Whennormalized to the signal from onput DNA in the ChIP reaction, andaveraged among experiments performed with different tumors, the bindingto the enhancer in AI tumors was only slightly lower than in AD tumors(see graph). In contrast, upon castration (ADc) AR binding to theenhancer and promoter decreased about 4-fold vs. AD tumors. There isstill evidence of some specific AR binding in ADc as the signals on theenhancer and promoter are above the IgG background. The residual bindingagrees with the cytology data in which some AR remains in the nucleuseven in ADc. Furthermore, the PSA levels do not decrease to baseline inADc but simply drop transiently as the tumor begins to transition to AI.

To analyze how preinitiation complexes respond to the presence andabsence of AR we measured binding of pol II and a representative generalfactor TFIIB (FIG. 28). We predicted that poL II would be distributedamong the promoter and downstream exon 5, and that it should disappearupon castration. However, the binding pattern was more complicated. PolII binding was observed at both the promoter and downstream exon 5 inthe AD tumors but its distribution was skewed. A scatter plot of theratio of pol II signal at exon 5 vs. the proximal promoter shows that inAD tumors the pol II is primarily at exon 5. We interpret the binding toexon 5 as an elongating polymerase. The signal is specific because it isfound only weakly at the enhancer and not in the intervening region.Binding by pol II to the enhancer was previously observed by Brown andcolleagues in their LNCaP cell culture study (Shang et al. 2002). Theauthors interpreted this phenomenon as evidence of DNA looping betweenthe enhancer and proximal promoter. Although we observe binding of polII to the enhancer, the binding to promoter and exon 5 is stronger

Remarkably, upon castration, pol II remained bound to the gene. However,as shown by the scatter plot, pol II was localized primarily at thepromoter versus exon 5 in four separate experiments. The AI tumors weremore complex; different experiments revealed more exon 5 signal and someless. This variation was in contrast to TFIIB, which was present atequal levels at the promoter in AD, ADc and AI in all experiments thatwe performed. Our interpretation of these findings is that thetranscription complex remains intact after androgen deprivation but agreater fraction of pol II is not actively transcribing in ADc vs. AD.This observation correlates well with the drop in the ADc imaging signalmeasured by CCD and the drop or plateau in serum PSA levels measured byELISA. The pol II position in Al tumors is more heterogeneous butmodestly skewed towards exon 5.

Visualizing the AD-AI Transition in Real Time

In a human, the failure of androgen deprivation therapy occurs graduallyover a period of time that can vary from weeks to years. The LAPC9models were originally adapted to an androgen-rich environment inimmunodeficient mice and then trained to grow in castrate or femalemice. We have found that the transition time in animals occurs morerapidly as the tumor grows larger. Typically we begin an experiment whena tumor reaches 0.5 cm but it grows to 1.5 cm within two or three weeks.Usually we have to sacrifice the animals before the transition occurs toadhere to the 1.5-cm tumor size limitation set by our institutionalanimal care guidelines. However, some individuals transition faster andin these we are able to monitor the transition in real time, as opposedto studying established AI tumors as we described above. The data belowillustrate a typical example of an animal that underwent the transitionprior to sacrifice (FIG. 29).

In this animal, we injected the AdTSTA virus, imaged the mice four dayslater, and then castrated the animal when the tumor reached 1 cm. Wefound that the PSA levels initially dropped 2.4-fold by day 10, six dayspost-castration, and then began rising again up to day 17, when we hadto sacrifice the animal because the tumor had reached the size limit.

Over this same time frame, the imaging signal was high on day 4 reacheda minimum by day 10 and then gradually rose again by day 17 (FIG. 29,top). ChIP and immunohistochemical analyses on the tumor from thesacrificed animal revealed that AR had resumed its predominantly nuclearlocation and was bound primarily to the PSA enhancer, while pol II wasfound predominantly in the elongating state at exon 5 (FIG. 29, bottom).In short, we show an example, where AR has adapted to theandrogen-deprived environment and resumed activity in the AI state asmeasured by imaging, immunohistochemistry and ChIP.

Discussion

Our study reports the development of a paradigm to study AR dynamicsduring prostate cancer progression. Cooled-CCD optical imaging is anemerging technology for monitoring transcriptional activity duringdevelopment, differentiation and disease (Contag et al. 2000; Contag2002; O'Connell-Rodwell et al. 2002). The ability to employ CCD imagingrequires potent reporter systems that can sensitively monitortranscriptional events in living animals. We employed anadenovector-based imaging cassette, which used the TSTA scheme tomeasure signals that impacted the AR-signaling pathway. The TSTA systemis robust and can dynamically monitor a specific androgen response invivo in the context of a tumor.

The goal of our study was to determine whether AR was functional in AIcancer. The natural response of an AD tumor to androgen deprivation istransition to an AI state. We found that the AdTSTA imaging system coulddetect the decreased AR activation caused by removing the systemicsource of androgen via castration. We further showed that the signalcould be restored in established AI tumors. Finally, we could monitorthe loss and gain of androgen responsiveness during a transitory periodwithin an animal. While this transition is significantly more rapid thanin a patient, it nevertheless demonstrates the ability of a potent butsensitive imaging system to monitor physiological changes that accompanytumorigenesis.

The imaging results paralleled several biological and mechanisticbenchmarks of AR function. The optical signals paralleled the halt (ordecrease) in rising PSA associated with the androgen depletion bycastration. We note that castration is unable to completely blockprogression of well-vascularized prostate xenografts, which ultimatelyreturn to the AI state. Also, the correlation was not exact, as the PSAlevels decreased on average 2 fold but the imaging signals decreasedover 10 fold within the same time frame. We do not yet understand thediscrepancy but it may simply reflect the relative half-lives of PSA vs.firefly luciferase. Alternatively, TSTA is an unusually sensitivereporter system designed to respond robustly to androgen. It may beresponding more dramatically to changes in the cascade of events thatcontrol AR activity. There is a delay in cell culture between theadministration of androgen and the appearance of a peak luciferasesignal. This delay is due in part to the time required to achievesteady-state activation of the reporter and also to the two step natureof the imaging amplification system. Nevertheless, within the timeframes used here, the trends of the imaging and PSA signals wereremarkably consistent.

The imaging signal also correlated somewhat with the cellulardistribution of AR, which was nuclear in AD and AI but became diffuse inADc animals. We note that the levels of AR drop 2-fold in ADc mice butrise an again in the AI state. Finally, the optical signal correlatedwith the AR binding pattern to the endogenous PSA enhancer, whichdecreased in ADc but increased again in AI cancer. While the cellularlocalization and enhancer binding of AR clearly cycled, the effects werenot absolute. As mentioned above, the PSA levels do not disappear buttransiently plateau or drop while the tumors adapt. This could explainthe residual AR signals in the ChIP experiments. We do not understandyet how the tumors adapt and this is the subject of much speculation andresearch in the field (Grossmann et al. 2001).

Indeed, one of the key issues in the prostate cancer field is whetherrecurrent cancer truly employs AR to drive its activity. Our study showsthat AR is functional in AI cancer. Although we have only studied thePSA gene we imagine that the resuscitation of AR activity is paralleledon the other genes used by AR in AD cancer growth. Several potentialmechanisms that might allow AR function in AI cancer have emerged overthe last few years.

Isolation of the AR gene from tumors and cell lines suggests thatcertain mutations may permit it to respond to alternate ligands and evenantagonists like flutamide (Barrack 1996). AR somatic mutations in theTransgenic Adenocarcimona of Mouse Prostate (TRAMP) model revealed acorrelation between reduced androgen dependence and mutations in regionsof AR known to interact with co-activators (Han et al. 2001).

Overexpression of AR has also been observed in AI cancer. For example,analysis of patient samples revealed that the AR gene is amplified in asubset (˜30%) of AI tumors (Visakorpi et al. 1995). However, steadystate levels of AR are also enhanced in patient samples, cell lines andxenografts even when the gene is not amplified (Gregory et al. 2001;Linja et al. 2001). It has been proposed that the augmented levels of ARor its co-activators can possibly drive AR function with castrate levelsof ligand, in part via the principles of mass action. It has also beensuggested that AR has adapted to utilize adrenal sources of androgensand possibly convert them to DHT. It is known that castration decreasescirculating DHT over 10-fold but smaller effects have been observed whenmeasuring DHT levels within the tumors (Grossmann et al. 2001).

Mitogen-activated protein kinase (MAPK) may impact AR activity. MAPK, amajor signaling pathway involved in cell proliferation, has been linkedto AR in numerous studies (Craft and Sawyers 1998; Abate-Shen and Shen2000; Elo and Visakorpi 2001; Feldman and Feldman 2001; Grossmann et al.2001; Arnold and Isaacs 2002). Elevated MAPK has been observed inadvanced Prostate cancer specimens from patients and in AI xenografts(Gioeli et al. 1999; Mellinghoff et al. 2002). Also, several receptortyrosine kinases or growth factors, which signal through MAPKs, activateAR-responsive reporter genes in an AI manner when overexpressed in cellculture (Craft and Sawyers 1998; Abate-Shen and Shen 2000; Elo andVisakorpi 2001; Feldman and Feldman 2001; Grossmann et al. 2001; Arnoldand Isaacs 2002). We note that the AR in LAPC9 has not undergonemutations but its levels are slightly enhanced in AI tumors and theLAPC9 AI models used here were previously shown to display enhanced MAPKlevels (Mellinghoff et al. 2002).

The relevance of AR in AI cancer is supported by several recent studies.Overexpression of murine AR from the probasin promoter in transgenicmice leads to development of high-grade prostatic intraepithelialneoplasia (PIN), which may be a precursor to prostate cancer (Stanbroughet al. 2001). Conversely, lowering AR levels by injection of ahammerhead ribozyme or antibodies targeted to AR reduces proliferationof AI LNCaP cells in culture (Zegarra-Moro et al. 2002). The effect ofAR in AI cell lines apparently requires DNA binding because the PSAenhancer requires some of its natural AREs to activate reporter genes inAI LNCaP cell lines, although other transcription factors alsocontribute (Yeung et al. 2000). Our data provide strong direct supportfor the notion that AR is indeed active and bound to its targetenhancers/promoters in natural tumor models of prostate cancer.

Although our data do not bear on the precise mechanism of AR function inAI cancer they provide evidence that AR is forming transcriptioncomplexes. As mentioned above, AR is bound to the PSA enhancer in ADtumors as measured by ChIP. We assume that this binding is paralleled onother AR-responsive genes but this point has not been firmlyestablished. Castration causes much but not all AR to be removed fromthe PSA enhancer but it apparently rebinds in the AI state. Similarly,pol II is primarily located on exon 5 in AD and in many AI tumors,possibly indicative of an elongating polymerase. The initial loss of DHTby castration causes pol II to relocalize mainly to the promoter in ADc.This phenomenon, along with stable TFIIB binding, suggests that thetranscription complexes probably remain intact in the absence ofandrogen, which may facilitate the reactivation of AR-mediatedtranscription during AI cancer.

Analysis of other genes such as á1-AT have also established that pol IIcan be bound with the GTFs in a quiescent state, prior to binding ofactivator (Cosma 2002). These data along with older studies of the heatshock locus in drosophila (Gilmour and Lis 1986) indicate that apre-poised pol II may provide a mechanism for maintaining promoteraccessibility during a transcriptionally inactive state. AR and pol IIare known to cycle on and off the promoter during gene activation inLNCaP cells (Kang et al. 2002; Shang et al. 2002). The peaks of AR andsubsequent pol II binding after androgen addition do not coincidesuggesting that AR can leave while pol II is engaging the promoter. Thisphenomenon may be analogous to what is occurring to pol II in ADctumors.

The chain of events that lead to the action of pol II at the PSApromoter remain to be firmly established in tumors due to the difficultyof precisely synchronizing cells in living animals. However, we haveestablished an experimental paradigm, where we can screen tumors viagene expression-based imaging followed by detailed molecular analysis(i.e., by ChIP). We have not delved into the precise co-activatorsdriving PSA or tumor progression because the genetic evidence linkingspecific co-activators to PSA expression is limited. Indeed, we havestrong evidence based on RNA interference that at least one co-activatorthought to allow AR function has no effect on the endogenous PSA gene.We are therefore systematically probing suspected co-activators by RNAinterference to validate their roles in AR-responsiveness.

On a technical note, we have concerns about GAL4-VP16 and fireflyluciferase toxicity. If the toxicity was an issue, we would predict thatinfected cells would die rapidly and the imaging signal would decay.However, under the conditions employed in our study we are able toobtain relatively persistent imaging signals in tumors within liveanimals over the course of the experiments, which can last up to amonth. Also, the virus is not unusually toxic in cell culture studies,where cells appear to thrive after virus infection. We note that ourability to employ low doses of virus in the animal studies may permit aless immunogenic response and enable us to transit the system intonon-SCID prostate models as well as transgenic animals. We are currentlyanalyzing the TSTA system in lentivirus-transformed lines and intransgenic animals. One limitation of the current study is that thetumor had to be palpable to inject the virus. Lentivirus transformedtumor cell lines are expected to be more stable than adenovirus-infectedcells and allow us to measure androgen-responsiveness at the earlieststages of prostate cancer growth. The use of low viral doses could alsomake possible the application of the system to therapeutic relatedresearch for early detection of cancer, a possibility we are currentlypursuing.

EXAMPLE 6

Safe and effective prostate cancer gene therapy requires high magnitudeand targeted expression in vivo. Targeted expression has the potentialto minimize unwanted side effect of therapy. We improved the activity ofnative prostate specific antigen (PSA) promoter and enhancer by nearly20-fold employing a chimeric enhancer manipulation strategy (Gene Ther2001, 8:1416). Moreover, in vivo application of the prostate specificchimeric viral vector resulted in the specific localization of humanprostate metastases in mouse xenograft model (Nat Med 2002, 8: 891). Toaccomplish efficient in vivo gene transfer we aim to improve theprostate-specific promoter activity to achieve magnitude comparable tostrong viral CMV promoter. We employed a two-step transcriptionalamplification (TSTA) approach, in which an artificial activator consistsof GAL4 and VP16 binds upstream and activates the expression of atherapeutic or reporter gene. The specificity of TSTA is governed by theuse of our modified prostate-specific promoter to express GAL4-VP16activator. We demonstrated that TSTA system exhibits higher activitythan CMV promoter but still maintained prostate specificity in cellculture transfections (Mol Ther 2002, 5:223). To optimize this systemfor in vivo applications, we characterized adenovirus vectors thatcontains the activator and the reporter component of TSTA in a single ortwo separate adenoviral vectors. In cell culture infection studies, weconfirmed that: 1) both single and separate TSTA system exhibit higheractivity than CMV, 2) both maintain androgen regulation, 3) theinfection with separate vectors showed higher androgen induction thansingle vector in prostate cancer cells due to lower basal expression inabsence of androgen and 4) high virus to cell ratio leads to diminishedcell specificity and androgen regulation. Our TSTA vectors expressfirefly luciferase gene which can be monitored in living mice byreal-time optical CCD imaging. In vivo expression of the single TSTAvector (AdTSTA-FL) exhibits cell selectivity and androgen regulation inprostate cancer xenografts and in murine prostate. Based on thesepromising results, we are actively investigating this TSTAtranscriptionally targeted approach in gene-based therapy and diagnosisfor prostate cancer.

EXAMPLE 7

Gene expression-based imaging should be a powerful tool to assess thereal time response of therapy. We have applied non-invasive opticalimaging to monitor HSV1 thymidine kinase suicide gene therapy inprostate cancer xenograft models. Here we report the utilization of anadenovirus-based two-step transcriptional activation (AdTSTA) system toexpress a potent HSV1 thymidine kinase variant (sr39tk) in the treatmentof LAPC-4 prostate tumors. The TSTA system consist of an enhanced PSApromoter driving the expression of a potent synthetic transcriptionalactivator GAL4-VP16 which in turn activates the expression of thetherapeutic gene (sr39tk) or reporter gene (firefly luciferase, FL). Forin vivo applications, AdTSTA-FL and AdTSTA-tk were generated to containthe activator and the reporter/therapeutic component in a single vector.The AdTSTA mediated expression is significantly higher than comparableCMV driven vector while maintaining exquisite cell selectivity andandrogen regulation. FL-based optical CCD imaging is a sensitive, facilemethod to sequentially monitor gene expression in living animals. Ourstrategy is to utilize FL mediated optical signal to measure therelative tk expression in the tumors during suicide therapy. We firstdemonstrated that co-infection of cells released from LAPC-4 xenograftswith equivalent dosage of AdTSTA-FL and AdTSTA-tk produced luciferaseactivity levels that are correlated with TK protein levels. LAPC-4 tumorbearing mice were divided into 3 therapeutic groups that receivedintratumoral injection of equal dosage of (i) AdCMV-RL+AdTSTA-FL(control group), (ii) AdCMV-sr39tk+AdCMV-FL (CMV group), or (iii)AdTSTA-sr39tk+AdTSTA-FL (TSTA group). 7 days post viral injection, theanimals were given daily intraperitoneal administration of 100 mg/kgganciclovir (GCV) for five days. Optical imaging just prior to GCVtreatment revealed that intratumoral signal in the TSTA group is morethan 1 order of magnitude higher than the CMV group. Serial imaging datarevealed a more precipitous loss of optical signal in the TSTA group incomparison to the CMV group in a 12 days period after GCV treatment.Detailed histological analysis revealed extensive central tumornecrosis, most dramatic in the TSTA over CMV group, and both treatmentgroups exhibit much greater cell death than the control group. In thisreport we demonstrate that TSTA system can mediate highly amplified andtissue-specific gene expression resulting in effective tumor cellkilling. Moreover, non-invasive optical CCD imaging is a useful tool tomonitor gene expression and therapeutic response.

EXAMPLE 8

We have previously reported the development of a Two-StepTranscriptional Amplification System (TSTA) to amplify fireflyluciferase (fl) reporter gene expression in vivo (Iyer et al, PNAS,2001). We now report the development and imaging of a novel transgenicmouse line that expresses fl transgene under the control of theProstate-Specific promoter and VP16 transcriptional activator.

Methods

The DNA fragment PSEBCVP2-G5-fl used for injection was isolated from theplasmid vector sequence by cutting with appropriate restriction enzymes.Transgenic mice were generated in FVB mice by standard techniques. Sevenfounder mice carrying the transgene were outcrossed with wild-typeanimals to yield F1 progeny. Tails from the F1 progeny were clipped at 2weeks of age and genotyped by PCR. The fl positive mice were imaged atdifferent intervals of time using a cooled Charge Coupled Device (CCD)camera and D-Luciferin as the substrate (3 mg per animal, injectedi.p.). The resulting bioluminescent signal was expressed asphotons/sec/cm²/steridian (sr).

Results

In male mice from F1 progeny, the expression of GAL4-VP16 dependent flgene is observed to be very strong and is primarily restricted to theprostate tissue (5×10⁶ p/sec/cm²/sr). In contrast, female transgenicmice show basal levels of fl expression (5×10⁴ p/sec/cm²/sr). Thissuggests the high androgen dependency of the prostate-specific promoter.Additionally, gene expression is also observed in the ears, feet andtail of male mice (1.7×10⁶-5×10⁶ p/sec/cm²/sr). Repetitive imaging overseveral months demonstrates persistence of the bioluminescent signal.The mice exhibit normal physical characteristics and development and donot show any deleterious effects from the transactivator.

Conclusion

We have developed a novel transgenic mouse line using the GAL4-VP16transactivator (driven by a prostate-specific promoter) to amplify theexpression of fl reporter gene and shown long-term tissue specificexpression of fl. Such transgenic mouse models when coupled with othertransgenic lines that spontaneously develop malignancies should play auseful role in the study of early events in cancer progression.

EXAMPLE 9

The progression of primary prostate cancer requires androgen and afunctional Androgen Receptor (AR). Treatments that reduce androgenlevels inhibit androgen dependent (AD) cancer progression andAR-responsive gene expression. Ultimately, the cancer regains thecapacity for aggressive growth in the androgen-independent (AI)environment and AR-responsive genes are re-activated. A key issue iswhether AR is directly mediating this AI response. Through extensivebiochemical studies we have identified the key AR responsive elements inthe Prostate Specific Antigen (PSA) enhancer. By combining enhancermodifications with two-step transcriptional activation (TSTA)approaches, we have crafted a robust, androgen-responsive opticalimaging system targeted to prostate cancer cells. Our “chimeric TSTA”employs a duplicated segment of the PSA enhancer core expressing GAL4fused to 2 VP16 activation domains. The resulting activators aretargeted to reporter templates bearing 5 GAL4 binding sites upstream offirefly luciferase. The entire TSTA imaging cassette is packaged into anAdenovirus for in vivo delivery into SCID mice bearing human prostatecancer xenografts. Using cooled charge-coupled device (CCD) imagingsystem we observe a robust, tumor-specific luciferase signal thatresponds to androgen depletion in live AD animal models. Remarkably, wehave also observed strong and persistent AR signaling in the AIxenografts. Following the imaging we analyzed the tumors using chromatinimmunoprecipitation assays. We identified AR association with theendogenous PSA enhancer and binding of RNA polymerase II, GeneralTranscription Factors (GTF) and the human mediator complexes to theendogenous PSA regulatory regions in both AD and AI tumors. These datacombined with immunohistochemical localization studies providemechanistic evidence that AR is fully functional in AI cancer. We havealso exploited the TSTA imaging system to detect cross-talk between theAR-pathway and the MAPK signaling cascade by switching the VP16activation domain with that of the MAPK-responsive ELK-1. Usingtransferrin-enhanced, liposome-assisted gene delivery, we haveintroduced the AdTSTA-ELK-1 into tumor xenografts through intravenousinjection and obtained signals in the distally located tumors. Currentlywe are assessing the combined MAPK-AR effects in AD and AI xenografts.AdTSTA and AdTSTA-Elk represents a series of tools for in vivo molecularimaging and therapeutic gene targeting in prostate cancer research.

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1. An expression vector comprising an effector nucleotide sequence,comprising a. a modified tissue specific enhancer sequence; b. a tissuespecific promoter sequence; and c. nucleotide sequence encoding achimeric transactivator operably linked to the modified tissue specificpromoter sequence, wherein the encoded transactivator comprises a DNAbinding domain and at least one viral transcription activation domain.2. A vector system comprising the expression vector of claim 1 and asecond expression vector comprising a reporter nucleotide sequence,wherein the reporter nucleotide sequence comprises a. multiple DNAbinding sequences; b. an Adenoviral early promoter sequence; and c. afirst heterologous gene sequence operably linked to the Adenoviral earlypromoter sequence.
 3. The expression vector of claim 1, wherein theeffector nucleotide sequence further comprises a second tissue-specificenhancer sequence.
 4. The expression vector of claim 1, wherein themodified tissue specific enhancer sequence is from blood, prostate,brain, lung, stomach, bladder, pancreas, colon, breast, ovary, uterus,cervix, liver, muscle, skin, or bone.
 5. The expression vector of claim1, wherein the modified tissue specific enhancer sequence is a modifiedProstate Specific Antigen (PSA) enhancer sequence.
 6. The expressionvector of claim 3, wherein the second tissue specific enhancer sequenceis a prostate specific enhancer sequence.
 7. The expression vector ofclaim 1, wherein the DNA binding domain is GAL4 or LexA.
 8. Theexpression vector of claim 1, wherein the viral transcription activationdomain is a VP16 or FKHR (forkhead).
 9. The expression vector of claim2, wherein the multiple DNA binding sequences comprise at least 1 and upto 5 DNA binding sequences.
 10. The expression vector of claim 2,wherein the multiple DNA binding sequences comprise GAL4, LexA, or PAX3.11. The expression vector of claim 2, wherein the Adenoviral earlypromoter sequence is from serotypes selected from a group consisting ofAd 1, 2, 3, 4, and
 5. 12. The expression vector of claim 2, wherein theAdenoviral early promoter sequence comprises an E1a, E1b, E2a, E2b, E3,or E4 sequence.
 13. The expression vector of claim 2, wherein theAdenoviral early promoter sequence comprises a TATA box sequence. 14.The expression vector of claim 2 further comprising a secondheterologous gene sequence operably linked to the first heterologousgene sequence.
 15. The vector of claim 1 which is a plasmid, cosmid, orphagemid.
 16. The vector system of claim 2 which is a plasmid, cosmid,or phagemid.
 17. The vector of claim 1 which is a bacterial vector,yeast vector, adenoviral vector, adenoviral-associated vector,lentiviral vector, or retroviral vector.
 18. The vector system of claim2 which is a bacterial vector, yeast vector, adenoviral vector,adenoviral-associated vector, lentiviral vector, or retroviral vector.19. A host-vector system, comprising the vector system of claim 2 or 14and a host cell.
 20. The host-vector system of claim 19, wherein thehost cell is a prokaryote or eukaryote.
 21. A method for expressing apolypeptide encoded by a heterologous gene sequence, comprising growingthe host vector system of claim 19 so as to express the polypeptide. 22.A method for expressing a polypeptide encoded by a heterologous genesequence in a subject, comprising implanting the host vector system ofclaim 19 in a subject so as to express the polypeptide.
 23. A method fordetecting expression of a polypeptide encoded by a heterologous genesequence in a subject, comprising administering the host vector systemof claim 19 to a subject under suitable conditions so as to express thepolypeptide encoded by the heterologous gene sequence and detecting thepolypeptide so expressed.
 24. The method of claim 22 or 23 furthercomprising administering to the subject an agent to induce expression ofthe polypeptide encoded by the heterologous gene sequence.
 25. Themethod of claim 24, wherein the agent is an androgen.
 26. The method ofclaim 23, wherein the detecting is effected by, positron emissiontomography (e.g., PET), single photon emission computed tomography,cooled charged coupled device (CCD) camera optical imaging, magneticresonance imaging, bioluminescent optical imaging, fluorescence opticalimaging, radionuclide imaging, or non-radionuclide imaging.